(CANCER RESEARCH 35. 1637 1642, July 1975]
The Role of Cell Division in the Malignant Transformation
of Mouse Cells Treated with 3-Methylcholanthrene1
Takeo Kakunaga
Department
of Tumor
Viruses. Research
Institute
for Microbial
Diseases. Osaka University.
SUMMARY
The requirement for cell division in the malignant
transformation of A31-714 cells, a subclone derived from
BALB/3T3, by 3-methylcholunthrene was investigated
using the property of the high susceptibility of this clone to
density-dependent inhibition of cell growth.
Treatment with 3-methylcholanthrene did not induce
transformation in a nongrowing population. However, the
cells treated with the carcinogen in a nongrowing state
showed a high transformation frequency near maximum
level when they were returned to the growing state soon
after treatment. About four cell generations were found to
be necessary for the development of cell transformation
after treatment with 3-methylcholanthrene.
Cells that were kept in a nongrowing state after carcino
gen treatment rapidly lost their ability to express transfor
mation even when they were subsequently returned to a
growing state. On the other hand, the cells that were allowed
one cell division soon after carcinogen treatment retained
their ability to produce transformed foci even after being
kept in the nongrowing state thereafter.
These results suggest that one cell generation is required
for the fixation of transformation and that several addi
tional cell generations are required for the expression of the
transformed state.
INTRODUCTION
It has been observed for a long time that young animals
and tissues that show a high growth rate have a high
susceptibility to chemical carcinogenesis. This finding seems
to be one of the important clues for elucidating the process
of chemical carcinogenesis.
In the studies on cultured cells, there have been 2 kinds of
findings suggesting a cell division requirement for cell
transformation by chemical carcinogens: (a) when altera
tion of clonal morphology was used as a criterion of cell
transformation. 1 to 3 days of growth after carcinogen
treatment were required for development of the transformed
state (4), and (h) when focus formation on the background
of monolayer of untransformed cells was used as an
indicator of cell transformation, the transformation fre
quency decreased in cultures treated at high cell density
1This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Japan.
Received December 16. 1974: accepted March 10, 1975.
JULY
Suita. Osaka. Japan
compared to cultures at low density (8, 19, 32). Recently, it
has been found that several cell divisions were required for
the appearance of transformed foci in cultures of A31-714
cells after 4NQO2 treatment (20). The present experiments
were designed to determine how many cell divisions are
required for the development of transformation induced by
a different type of carcinogen, MCA, and to determine what
steps in cell transformation require cell division. MCA was
chosen from several chemical carcinogens that have been
shown to transform A31-714 cells quantitatively because of
its low cytotoxic effects on A31-714 cells (19). This
eliminated both the complexity in interpreting the results
and the restriction on some experimental approaches due to
the potent cytotoxic effect of 4NQO (22).
MATERIALS
AND METHODS
Chemicals. MCA was purchased from Nakarai Chemical
Company (Kyoto, Japan), dissolved in dimethyl sulfoxide
immediately before using, and added to culture medium at a
final concentration of 1.0 ¿/g/mlfor 24 hr. This concentra
tion of MCA is the midpoint of the range that gives a linear
dose-response curve for cell transformation and that does
not cause significant change in plating efficiency of
A31-714 cells. The control cultures, which received di
methyl sulfoxide alone, did not produce any transformed
foci under the conditions used for assay of transformation.
Cell Cultures. A31-714 cells ( 19), a subclone derived from
BALB/3T3, were cultured in 60-mm plastic dishes (Falcon
Plastics, Oxnard, Calif.) containing 5 ml Eagle's minimum
essential medium supplemented with 10%calf serum, unless
otherwise specified. To prepare the culture medium con
taining 30% serum, dialyzed calf serum was used because
calf serum contains the dialyzable cytotoxic factors that
damage cultured cells at this high concentration. The
medium was changed 2 or 3 times a week. The cultures were
incubated at 37°in a CO2 incubator. In some experiments,
depleted medium was used in place of fresh medium so that
the cells would not grow beyond their saturation density.
(This author found that cells growing in fresh medium in
most cases will temporarily grow a little beyond their
saturation density; unpublished observation). The term
"depleted medium" is used to denote the medium in which
the factors present in fresh serum that release cells from
density-dependent inhibition of cell growth are depleted by
2The abbreviations used are: 4NQO, 4-nitroquinoline I-oxide; MCA,
3-methylcholanthrene.
1975
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1637
T. Kakunaga
previous exposure of the medium to confluent cultures.
However, the ability to support the growth of subconfluent
cultures remains. Depleted medium containing 10 or 30%
calf serum was prepared by exposing the culture medium
containing the corresponding concentration of serum for 3
days to confluent cultures of A31-714 cells that had been
cultured in the culture medium supplemented with 10 or
30% calf serum, respectively. Depleted medium was stored
at -20° until use, after eliminating cell debris by centrifuging at 1500 rpm for 10 min.
Assay for Transformation. Assay for cell transformation
by MCA and the phenotype of transformed cells has
previously been described in detail (19). In brief, 24 hr after
plating, the cells were exposed for 24 hr to medium
containing 1 /xg MCA per ml, then they were washed twice
with Dulbecco's phosphate-buffered saline [NaCl (8
RESULTS
Requirement for Cell Division for the Development of Cell
Transformation. The average number of cell generations
required to attain saturation density was calculated by
assuming that all the cells in cultures underwent the same
number of cell divisions before the saturation density of 33.9
x IO4cells/plate (1.2 x IO4cells/sq cm) was attained. As
shown in Table 1, when A31-714 cells were seeded at 1 x
IO4cells/plate, the cultures became confluent about 9 days
after seeding, at which time the percentage of cells synthe
sizing DNA markedly decreased. When the cells were
treated with MCA at different times after the seeding, the
transformation frequency per treated cell decreased. This
decrease was correlated with the time after the seeding, the
increase in the cell density, and the decrease in the cell
g/liter), KC1 (0.2 g/liter), Na,HPO4 (1.15 g/liter), KH2
generations required to attain saturation density.
To determine whether the cells treated in a nongrowing
PO 4 (0.2 g/liter), CaCl2 (0.1 g/liter), and MgCl2 6H2O
(0.1 g/liter), pH 7.2], and incubated in the carcinogen-free state are able to produce transformed foci when they are
medium. Twenty-nine days after the treatment the cultures returned to the growing state, and to determine how many
were stained with Giemsa, and the transformed foci (which cell divisions are necessary for development of transforma
were deeply stained against a lightly stained background of tion, confluent cultures were treated with MCA for 24 hr
untransformed cells) were scored. The loss of density- and immediately seeded at 6 different seeding levels using
dependent inhibition of growth, which led to the formation carcinogen-free depleted medium (see "Materials and
of transformed foci, was used as the criterion of the Methods"). The depleted medium was used during the first
10 days of incubation so that the cells would not divide after
transformed phenotype.
Cell Counts. The number of cells per dish was determined reaching saturation density. Medium change with fresh
by hematocytometer counts of suspended cells or by medium did not induce a remarkable increase in cell number
counting the number of cells in limited areas of the dish over saturation density once the monolayer cells had been
formed. Six levels were chosen so as to give an approxi
under a microscope.
Percentage of the Cells Synthesizing DNA. Cells were mately known number of cell divisions before saturation
exposed to [3H]thymidine (0.1 ¿iCi/ml)for 30 min or 24 hr. density was attained. As shown in Table 2, most of the cells
Then the cells were fixed with methanol and washed 3 times in the cultures inoculated at 1 to 16 x IO4cells per plate
with cold 5% perchloric acid. The percentage of the cells synthesized DNA within 24 hr and all cultures reached
saturation density by the 10th day after plating. On the
labeled was determined autoradiographically (21).
Transformation
frequencies
Table 1
in cultures treated with MCA
at different
limes after seeding cells
A3I-7I4 cells were seeded at an inoculum size of 10' cells/plate and treated with MCA at
different times after the seeding. The number of cells per plate and the percentage of cells
incorporating [3H]thymidine at the time of MCA treatment.
Initiationof
24-hrtreatment
no.of
cellgenerations
with MCA
required
to attain
cells
(days after
of
saturation
incorporating
of
seeding
cells x
[3H]thymidine'95N.T.'796592No.
lO'/plate"0.952.59.417.630.633.9Av.
density"5.13.71.90.90.10%
foci/plate1*7.1
cells)1357912No.
frequency/
10streated
cells75
±0.9»9.9
±940
1.17.3
±
±57.8
0.81.0
±
0.71.8
±
±0.51.4
±0.30.46
±0.40.9
±0.10.27
±0.3Transformation ±0.08
" Averages of values in 3 plates.
" Assuming that all the cells have undergone the same number of cell divisions before the
saturation density was attained.
' Averages of values in 2 plates. Determined autoradiographically on the cultures exposed to
[3H]thymidine for 24 hr beginning 12 hr before and ending 12 hr after the indicated time.
" Scored 29 days after the MCA treatment.
' Mean ±S.E. for 8 to 20 plates.
' Not tested.
1638
CANCER
RESEARCH
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Cell Division and Transformation by MCA
Table 2
Effect of cell inoculum si:e on the Iransformation frequencies of A31-714 cells treated with MCA
A3I-714 cells in confluent cultures were treated with MCA for 24 hr, washed twice with Dulbecco's
phosphate-buffered saline, suspended, and then seeded at various inoculum sizes using depleted medium.
Number of transformed foci was scored 29 days after the treatment.
ofcells
xlOVplate1
no. of
cell
generationsrequiredto
cellsincorporating[3H]thymidine'89.589.094.293.879.94.72.0No.
attainsaturationdensity"5.03.93.01.91.0O.I0%
treatedcells57
foci/plate5.7
of
afterseeding"1.02.14.18.416.531.932.0Av.
day
lOVplate12481632Confluent
No.
cellsinoculatedx
of
0.5"7.0
±
±535
±34.8
0.61.9
±
1.03.0
±
0.42.4
±
0.40.69
±
0.31.1
±
±0.190.13
±0.30.4
±0.060.06
0.20.2
±
±0.04
±0.1Transformationfrequency/10s
culture1No.
" Averages of values in 2 to 3 plates.
" Assuming that all the cells have undergone the same number of cell divisions before the saturation density
was attained.
' Averages of values in 2 plates, determined autoradiographically.
'' Mean ±S.E. for 7 to 11 plates.
' Confluent cultures were not transferred after MCA treatment.
other hand, the cultures inoculated with 32 x IO4 cells
showed a low percentage of cells synthesizing DNA during
the 24 hr after seeding, and most cells in these cultures did
not divide. The cells treated with MCA in the nongrowing
state showed almost the maximum transformation fre
quency when they were replated at an inoculum size of 1 x
10*cells/plate so that they could divide more than 4 times.
On the other hand, transformation frequencies were low in
the cultures reseeded at the higher inoculum sizes. That the
cultures replated at the inoculum size of 32 x 10*cells/plate
also gave a very low transformation frequency indicates that
trypsinization of cells or transfer of culture alone did not
influence the development of the transformed state. These
results suggest that about 4 cell generations are necessary
for the development of the transformed state after MCA
treatment.
Requirement for Cell Division for the Fixation of the
Transformation. To determine what steps in cell transfor
mation require cell division and how long the treated cells
maintain their abilities to produce transformed foci in the
nongrowing state, the cells, which were treated with MCA
at the confluent state and subsequently allowed either no or
1 cell division, were returned to the growing state at various
times after being kept in the nongrowing state. Two
procedures were chosen as a means of inducing only 1 cell
division in the cells in confluent cultures; one procedure was
the transfer of confluent cultures at the split ratio of 1:2
(Chart 1), and the other was elevation of serum concentra
tion in culture medium to 30% from the usual 10% (Chart
2). In these experiments the depleted medium was used.
Both procedures induced about 1 semisynchronized cell
division and consequent arrest of cell growth as can be seen
in the charts.
First, confluent cultures were treated with MCA for 24 hr
and washed. Then the cells were suspended by trypsinization
100
S
E
n
=
1
23456
Days After Plating
Chart I. Number of cells per plate and percentage of cells incorporat
ing ['Hjthymidine at different times after rcplating confluent culture at the
split ratio of 1:2 using depleted medium. [3H]Thymidine was added to
culture medium for 24 hr beginning 12hr before and ending 12 hr after the
indicated time. Each value represents average in 2 or 3 plates.
and seeded at 2 different seeding levels. The 1st group of
cells was seeded at an inoculum size of 32 x IO4cells/plate
and the 2nd group at 16 x IO4cells/plate, so that the former
group of cells could not grow and the latter group divided
only once before reaching confluence. Then, at intervals,
cultures were trypsinized and replated at 1:16 dilution in the
former group and at 1:8 dilution in the latter group so that
all the cells could divide 4 times after MCA treatment.
Chart 3 shows the transformation frequency obtained as a
function of the time elapsed between the MCA treatment
and the replating of cells. The cells of the 1st group, which
were kept in a nongrowing state after MCA treatment, lost
their ability to produce transformed foci as a function of
time. On the other hand, the cells of the 2nd group, which
were allowed 1 cell division soon after MCA treatment,
retained their ability to be transformed even after being
JULY 1975
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1639
T. Kakunaga
ent state under the usual culture conditions. On the other
hand, the lower transformation frequency in the cultures
that were maintained in a confluent state throughout the
experiment can be ascribed to the poor expression of the
transformation, which had been fixed in a small number
of cells.
Second, confluent cultures were treated with MCA,
washed, and divided into 2 groups; one was maintained
without any changes in usual culture conditions, and
the other was exposed to the fresh medium containing 30%
calf serum for 24 hr and then cultured in the depleted
medium containing 30% calf serum. The former group of
012345
cells
did not undergo division while most cells in the latter
Days After Increase in Serum Concentration
group divided once before attaining the new saturation
Chart 2. Number of cells per plate and percentage of cells incorporat
density. The cultures were then transferred at a split ratio of
ing [3H]thymidine at various times after increasing the serum concentra
1:16 in the former group and 1:8 in the latter, so that all the
tion from 10 to 30"(. [3H]Thymidine was added to culture medium for 30
cells could divide more than 4 times in total after MCA
min. Depleted medium was used. Each value represents average in
treatment. The results shown in Chart 4 were almost the
duplicate plates.
same as the 1st experiments, indicating that different
conditions of induction of cell division gave similar results.
These results suggest that 1cell division within 1 or 2 days
after MCA treatment is required for the Fixation of the
transformation.
DISCUSSION
The results described here suggest that 1 cell division is
required for the fixation of transformation and that this
division must occur within 1 or 2 days after MCA treat-
i
3
Time of Holding the Cells m Non Growing
State After MCA Treatment (days]
Chart 3. The ability of the cells to be transformed as a function of time
of holding the cells in a nongrowing state after MCA treatment. Confluent
cultures of A31-7I4 cells were treated with MCA for 24 hr, washed twice
with Dulbecco's phosphate-buffered saline, suspended, and divided into 2
groups. The cells of the 1st group (O) were seeded at an inoculum of 32 x
10*cells/plate, and those of the 2nd one (D) were seeded at 16 x 10' cells
using depleted medium. The cultures were then transferred at the indicated
time at 1:16 dilution in the former group and at 1:8 dilution in the latter
group using fresh medium. The number of transformed foci was scored 29
days after the final seeding of cells. The value at zero time is that obtained
in a culture seeded at an inoculum of 2 x 10*cells/plate immediately after
MCA treatment. Each value represents mean ±S.E. for 7 to 11 plates.
E l
01234
56
Time of Holding the Cells in Non-growing
State After MCA Treatment
kept in the nongrowing state for 5 days thereafter. The ob
served low-frequency transformation
in MCA-treated
cultures that were kept in a nongrowing state for 5 days and
returned toa growing state thereafter seems to be due to in
complete inhibition of cell division in confluent cultures.
The transformation fixed in a small number of cells by this
mechanism would be fully expressed by the subsequent 4
cell generations. As shown in Tables 1 and 2 and Charts 1
and 2, a small population of cells divided even in a conflu
1640
(Days)
Chart 4. The ability of the cells to be transformed as a function of the
time of holding the cells in a nongrowing state after MCA treatment.
Confluent cultures of A31-7I4 cells were treated with MCA for 24 hr,
washed twice with Dulbecco's phosphate-buffered saline, suspended, and
divided into 2 groups. The cells of the 1st group (O) were maintained
without changes in culture condition, and those of the 2nd (D) were
cultured in the depleted medium containing 30% calf serum. The cultures
were then transferred and the number of transformed foci was scored as
described in the legend to Chart 3. Each value represents the mean ±S.E.
for 7 to 10 plates.
CANCER RESEARCH
VOL. 35
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Cell Division and Transformation by MCA
ment. About 3 additional cell divisions seem to be necessary
for the expression of the transformed state. These results
were similar to those obtained with 4NQO (20). It has been
reported that the morphological transformation of hamster
embryonic cells required 1 or 2 days for the expression after
treatment with polycyclic hydrocarbon (4) and 2 cell
generations for the fixation after X-irradiation (5, 6). The
differences in the number of cell generations necessary for
fixation or expression between these results and ours may be
due to the differences in either the criteria used to score
transformation or type of cells used.
The number of cell generations calculated in this experi
ment is, indeed, a crude index, especially because the
doubling rate of the freshly transformed cells is in question.
Preliminary experiments, however, showed that treatment
of the cells with MCA for 24 hr at the concentration of 1.0
//g/ml did not cause remarkable change in the doubling time
and that there was no difference in the doubling time in the
subconfluent state between the untransformed cells and the
transformed cells when it was determined soon after the
isolation of the transformed cells from transformed foci.
The requirement for cell division soon after carcinogentreatment for the fixation of transformation and the loss of
fixation during a nongrowing period can be explained by
assuming that carcinogen-induced damage is converted into
a stable, replicable form only by means of cell division
before repair occurs (22). This hypothesis is similar to that
used to explain some mutagenesis in microorganisms (23,
39, 40), although alternative explanations for our observa
tions, such as damage to the DNA replication machinery
and epigenetic change, are also possible. It has already
been demonstrated that A31-714 cells exhibit the excision
type of repair in a nongrowing state, i.e., induction of un
scheduled DNA synthesis (18), excision of carcinogen
adducts to DNA (17), and excision of thymine dimers after
UV irradiation (unpublished data). Recently, it has been
reported that synchronized mouse cells showed maximum
transformation frequency when they were treated in late G[
or S phase with /V-methyl-jV-nitro-./V-nitrosoguanidine or
7,12-dimethylbenz(a)anthracene-5,6-oxide (3, 25, 29). This
finding may be related to our data, although it is unknown
whether the mouse cells that were used in those experi
ments exhibit the excision type of repair in a nongrowing
state or whether the chemicals used in those experiments
cause the same type of damage to cells as 4NQO and
MCA in this study.
The necessity for several cell divisions for the expression
of the transformed state may be due to the polyploidy of the
cells used. An alternative explanation is that cell division
may result in dilution or destruction of some component
that inhibits the expression of the transformed state. The
number of cell divisions required will be dependent on the
criteria used as diagnostic indices of the transformed
phenotype. Agglutinability by concanavalin A in 3T3 cells
infected with SV40 has been reported to be manifested after
1 cell generation (1).
Results similar to those described here have been reported
with SV40 transformation of 3T3 cells (15, 37, 38). Cell
division soon after infection with DNA tumor viruses seems
to be necessary for the integration of viral genome into host
JULY
DNA (7, 11, 14, 34, 36), which would presumably be quite a
different process from that of fixation of cell transformation
by chemicals.
The finding that both of the steps in cell transformation.
Fixation and expression, require cell division seems to af
ford a possible basis for the interpretation of the following
in vivo findings: (a) tissues, organs, and animals that contain
cells with a high growth rate are more susceptible to tumor
incidence by carcinogens (2, 13, 24, 26-29, 33); (b) most
carcinogens have cytotoxic effects that would result in the
induction of cell division; (c) promoters of carcinogenesis
stimulate cell proliferation (9, 10, 12, 16, 30, 35); and (d) the
promotion of tumor incidence is observed when the-pro
moter is repeatedly applied beginning sometime after
carcinogen injection, and this promoting activity is further
enhanced by a single additional application of promoter
before the carcinogen injection (12, 16, 30, 31, 35).
ACKNOWLEDGMENTS
I am very much indebted to Dr. C. Wesley Dingman, Dr. Rufus S. Day,
III, and Dr. James P. Whitlock for reviewing the manuscript and to Dr.
Sohei Kondo. Dr. Juntaro Kamahora, and Dr. Kumao Toyoshima for
valuable suggestions and discussions. Thanks are also due to Barbara
Heifetz for preparation of the manuscript.
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CANCER RESEARCH
VOL. 35
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The Role of Cell Division in the Malignant Transformation of
Mouse Cells Treated with 3-Methylcholanthrene
Takeo Kakunaga
Cancer Res 1975;35:1637-1642.
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