[CANCER RESEARCH 31, 59—65, January 1971]
Effect of Insulin on DNA Synthesis and DNA Polymerase
Activity in Organ Culture of Rat Mammary Carcinoma,
and the Influence of Insulin Pretreatment and of Alloxan
Diabetes1
J. C. Heusonand N. Legros
Department of Internal Medicine2 and Laboratory of Clinical Investigation, Institut Jules Bordet, Centre des Tumeurs de l'UniversitéLibre de
Bruxelles, Brussels,Belgium
SUMMARY
As previously reported , 7,1 2-dimethylbenz(a)anthracene
induced mammary carcinomas of the rat are usually insulin
dependent for cell proliferation in organ culture, although a
few proliferate readily in the complete absence of insulin. This
problem was further investigated by studying the effect of
insulin on DNA polymerase in the cultured explants.
It was found for most tumors that DNA synthesis and DNA
polymerase activity ran a closely parallel time course. Stimula
tion of DNA synthesis was delayed in onset and was accom
panied by a concomitant rise in DNA polymerase activity. In
tumors with insulin-independent DNA synthesis, DNA polym
erase was also insulin independent. These observations support
our earlier interpretation
that the stimulating property
of insulin is not merely a permissive effect, mediated through
energy-yielding
reactions,
but
rather
involves
activation
or
induction of enzyme systems responsible for DNA synthesis.
The level of the DNA-synthesizing process at onset of
culture varied markedly from tumor to tumor, even in the
same rat. It ranged from low to highly activated. The activated
state, which was most frequently encountered, would seem to
result from an “overresponsiveness―
of the process to stimula
ting factors in vivo. The fact that inactivation invariably
occurred after induction of alloxan diabetes, together with
other lines of evidence, suggest that insulin may be one of
these factors.
Hypersensitivity to stimulating factors and insulin independ
ence possibly represent successive steps toward escape from
the normal growth-regulating mechanisms. Loss of insulin
dependence, as revealed in culture, appears to accompany the
specific ability of the tumor to grow in diabetic rats.
@
was
more directly
related to the
control of DNA synthesis. Another interesting feature was
that, in addition to tumors with insulin-dependent cell prolif
eration, others displayed an intense mitotic activity occurring
spontaneously and independently of the presence of insulin in
vitro. These two types of tumors appeared to be identical on
histological examination. The loss of insulin dependence in the
second
normal
plastic
The
related
type
was thought
to result
from
alteration
of the
growth-regulating mechanisms inherent in the neo
process.
contention that insulin seemed to act on processes
to the control of DNA synthesis led us to study its
effect on DNA polymerase
activity in the tumor explants.
This
enzyme system was selected because, as reported by Lock
wood et aL (13), initiation of DNA synthesis by insulin in the
normal epithelial cells of mammary explants from mature mice
involved emergence of DNA polymerase activity in these cells.
The same relationship between DNA synthesis and activity of
this enzyme
was observed
by other
authors
in various mam
malian tissues subjected to the proper stimulus (2, 3, 14, 16,
21).
The present work studies the effect of insulin on DNA
synthesis and DNA polymerase activity in explants of both
insulin-dependent
and insulin-independent
rat mammary
tumors. It also examines the effect of insulin pretreatment
and
of alloxan diabetes in vivo on the properties of the tumors in
culture. The purpose of these studies was to investigate the
mechanism of the growth-promoting
effect of insulin on the
possible
In earlier studies (6, 7), it was demonstrated that insulin
stimulated cell proliferation in explants of carcinogen-induced,
work
seemed likely to act on processes
rat mammary tumor in vitro and to derive conclusions on the
INTRODUCTION
I This
hormone-dependent mammary carcinomas of the rat in organ
culture. It was also shown that this effect was not mediated
through an increase in glucose uptake and utilization by the
explants and, therefore, appeared not to be a simple permissive
effect bearing on energy-yielding reactions. Instead, insulin
supported
in
part
by
Contract
role of insulin on the control
of their growth in vivo.
MATERIALS AND METHODS
EURATOM
ULB-PISE 026-634-BIAC
and by a grant of F&lération
Animals. Mammary tumors were induced in Sprague-Dawley
Belgo-Luxembourg eoise des Industries du Tabac to the European
female rats by a single feeding of 7,l2-dimethylbenz(a)Organization for Research on Treatment of Cancer.
anthracene at age 50 days (9). Tumors studied in culture were
This department
is affiliated
with the European
Organization
Research on Treatment of Cancer.
Received May 11, 1970; accepted September 23, 1970.
JANUARY
for
obtained both from untreated rats and from rats receiving one
of the following treatments. One group of rats received 2.5 i.u.
1971
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59
J. C. Heuson and N. Legros
of Novo-Lente insulin (Novo A/S, Copenhagen, Denmark)
per 100 g of body weight s.c. daily, except on Sundays, for
varying periods of time before tumor excision. In addition,
they were given a 10% glucose solution as drinking fluid in
order to protect them from lethal hypoglycemia. Another
group was made diabetic by a single i.p. injection of alloxan
(12 mg/lOO g of body weight). For reduction of mortality in
the latter group, insulin replacement therapy was started on
the following day and given in decreasing doses for several
days. The day following cessation of insulin administration
was taken as Day 0 of diabetes. Rats were considered diabetic
when the blood serum glucose was in excess of 350 mg/lOO ml
(glucose oxidase method, glucostat reagents, Worthington
Biochemical Corp., Freehold, N. J.).
Culture Method. Explants of freshly excised tumor tissue
were prepared for organ culture by placing 12 fragments of
about 1 cu mm on a Millipore filter disc. Each disc was main
tamed
at the
surface
of 2 ml of the
chemically
defined
Medium 199 enriched with glucose (0.25 g/l00 ml) for culture
periods of 2 to 4 days at 37°.The gas phase was 95% 02 and
5% CO2 . The medium was changed daily and contained bovine
crystalline insulin (24.3 i.u./mg, Calbiochem, Los Angeles,
Calif.) at a concentration of 40 j.zg/ml. Control explants were
cultured
in parallel without
insulin.
Assay for DNA Synthesis and DNA Polymerase Activity.
DNA synthesis and DNA polymerase activity were measured
simultaneously in the explants. The term explant is used here
as a unit to designate a group of 12 fragments placed on a
Millipore filter disc. The determinations
were carried out on 3
to 4 replicate explants.
DNA synthesis was determined after a 4-hr exposure to 0.5
j.zCi/ml of thymidine-3H (specific activity, 1.15 Ci/mmole,
Calbiochem, Los Angeles, Calif.). The explants were washed 4
times in Earle's base containing 0.1% glucose and mM
thymidine, blotted, weighed, homogenized at 0°in 0.5 ml of a
solution containing 0.25 M sucrose, 12 mM Tris-HC1 (pH 7.8),
and 6 mM KC1, and then centrifuged at 800 X g for 10 mm.
The sediment which was found to contain more than 98% of
the acid-insoluble
radioactivity
was resuspended
in 0.5 ml of
(1 .zCi) of dATP-3 H (specific activity, 4.8 to 15.1
Ci/mmole; Schwarz BioResearch, Inc., Orangeburg N. Y.);
100 pg of heat-denatured salmon sperm DNA; and 0.05 ml of
enzyme preparation (supernatant traction). However, the basic
concentration of the 4 dNTP's (dNTP X 1) was increased in
the reaction mixture, for reasons explained below, usually by a
factor of 6 (dNTP X 6) or, when enzyme activity was low, by
a factor of 2 (dNTP X 2). The reaction was carried out at 37°
for 60 mm and was arrested by adding 1 ml of a 0.5% casein
solution containing 333 mj.zmoles of ATP and 1.25 ml of cold
N PCA. The procedure described by Lockwood et aL (13) for
washing the acid precipitate was not used here because it gave
high radioactivity values in the blanks. It was replaced by a
procedure based on a method described by Younger et aL
(21). The precipitate was centrifuged at 2000 rpm for 5 min in
the cold. The sediment was dissolved at 0°in 0.5 ml of 0.5 N
NaOH. The resulting solution was completed by adding, in
rapid succession@ 0.5 ml of water containing 666 mpmoles of
ATP and 1 ml of N PCA. The procedure was repeated 3 times.
It yielded very low blanks and a high recovery of the
heat-denatured DNA. The final precipitate was washed twice
with ethanol:ether (3: 1), dissolved in Hyammne, and counted
in a liquid scintillation spectrometer.
The results were
corrected for quenching and expressed as jipmoles of dATP or
dpm/mg of tissue, wet weight, in the enzyme preparation.
The reason for increasing 2- to 6-fold the concentration of
the 4 dNTP's in the reaction mixture over that used by Lock
wood et aL ( 13) for the mouse mammary tissue is shown in
Chart 1. The substrate concentration dNTP X I yielded, for
this tumor-enzyme preparation, a reaction velocity below
Vmax and nonlinear kinetics during incubation periods of 60
mm. Concentrations3 or 6 timeshigher(dNTPX 3 or X 6)
gave maximal velocity and linear kinetics.
Omission of the dNTP's from the reaction mixture pro
duced a decrease in dATP-3 H incorporation. The decrease was
20% when dCTP was omitted,
mM thymidine. The acid-insoluble material was precipitated
by adding 1 ml of N PCA.3 The precipitate was washed twice
with 2 ml of N PCA and twice with ethanol:ether (3: 1). It was
dissolved in 1 ml of Hyamine and counted in a liquid
scintillation spectrometer. The results were corrected for
quenching and expressed as dpm/mg of tissue, wet weight.
DNA polymerase activity was estimated by a modification
of the methods described by Bollum (1) and Lockwood et aL
(13). The supernatant fluid of the 800 X g centrifugation was
recentrifuged in the cold at 90,000 X g for 60 mm or at
200,000 X g for 30 mm. The supernatant fluid was used as the
enzyme preparation.
The basic reaction mixture for assay of DNA polymerase
activity
contained
3The abbreviations used are: PCA, perchloric acid; dNTP, deoxy
nucleoside triphosphate.
60
12
dWTPx3
41C
IE
(Li
4
(Li
I
-a
0
0.
4
z
in 0.25 ml: 20 j.tmoles of Tris-HC1 buffer
(pH 7.4); 2 jimoles of MgC12; 0.25 @.tmoleof 2-mercapto
ethanol; 0.25 jimole of EDTA; 30 mpmoles each of dGTP,
dCTP and TTP; 15 mj.@molesof dATP; 0.2 1 to 0.07 mj.imole
50% when dGTP or TTP was
omitted, and 80% when all three were omitted. This shows
that the activity is mainly composed of the replicative enzyme
(11).
0
TINE
OF INCUBATION
( NIN)
Chart 1. Kinetics of DNA polymerase activity (enzyme preparation
obtained from pooled tissue of 3 tumors) at 3 different substrate con
centrations.
Results expressed
as @tjimoleof dATP-3 H incorporated
into
primer DNA/mg of tissue in the enzyme preparation.
CANCER RESEARCH VOL. 31
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DNA Polymerase in Cultures ofMammary
Carcinoma
E
E
V
‘I,
‘F,
(U
I
z
>.
“a
4
z
0
2
4
@:i:@
DURATIONOFCULTURE
(DAYS)
®DURATION
OFCULTURE
(DAYS)
Chart 2. Effect of insulin on DNA synthesis and on DNA polymerase
activity (substrate concentration, dNTP X 6) in explants of a tumor
from an untreated rat. Low initial level of the 2 parameters (inactive
Chart 3. Effect of insulin on explants of another tumor from an
untreated rat. High initial level of DNA synthesis and of DNA polym
erase activity (activated tumor). Conditions and symbols as in Chart 2.
tumor).
DNA synthesis
expressed
as dpm of thymidine-3
H incorporated
into cell DNA (4-hr exposure)/mg of tissue. Results of DNApolymerase
assay expressed as dpm of dATP-3 H incorporated into primer DNA
(60-mm incubation)/mg tissue in the enzyme preparation. DNA
synthesis in the presence of insulin, o—o; in the absence of insulin,
.—..
DNA
polymerase
activity
in
the
presence
of
insulin,
@-- -a', in
the absence of insulin, @-
tumors, daily measurements disclosed an irregular time course
of DNA synthesis in the insulin-treated explants, suggesting
that partial synchronization of the mitotic activity may have
occurred.
Chart 4 illustrates the behavior of still another type of
insulin-dependent
RESULTS
The effect of insulin on DNA synthesis and on DNA polym
erase activity in culture of 2 to 4 days was studied in a series
of 45 tumors. Sixteen of these were obtained from untreated
rats, 13 were from insulin-pretreated rats, and 16 were from
alloxan-diabetic rats. It was found that a majority of the
tumors (36 tumors) needed insulin for DNA synthesis, whereas
8 were insulin independent
and 1 showed
a negligible
rate of
DNA synthesis. Since the stimulating effect of insulin was
delayed in onset (cf. below), tumors were defined as insulin
dependent when insulin produced a significant increase in
DNA synthesis on Day 2 of culture.
close
Insulin-dependent
Tumors
Untreated Rats. Fourteen of the I 6 tumors in this group
were insulin dependent. Chart 2 demonstrates the effect of
insulin on such a tumor during culture. DNA synthesis and
DNA polymerase activity ran a closely parallel time course.
Their values were low at onset of culture and were unaffected
by insulin during the first 4 hr. Thereafter, they rose sharply in
the presence of insulin, while they remained low in its absence.
The lack of effect of insulin during the first 4 hr of culture
was a constant finding in all 12 insulin-dependent tumors
where this particular point was investigated. In view of this
observed constancy, the initial measurements were carried out,
in the other tumors, only on the control explants.
The insulin dependence expressed itself in several different
ways, illustrated in the following charts.
Chart 3 shows the result of a culture experiment performed
with another insulin-dependent tumor. The results differ from
those of Chart 2 in that the initial values of DNA synthesis and
of DNA polymerase activity were high. The effect of insulin
was only to maintain these high values throughout culture. In
the absence of insulin, they dropped abruptly. In some
JANUARY
tumor;
2 cases of this type were found
in
the group of untreated rats. DNA synthesis and DNA polym
erase activity had high initial values, followed by a sharp
drop both in the presence and absence of insulin. In this
tumor, the time course of DNA polymerase activity displayed
particular features that will be described below.
Thus, the level of DNA synthesis and of DNA polymerase
activity at onset of culture, which may be assumed to reflect
the state of the tumor in vivo at the time of excision for
culture, varied among tumors between the extremes repre
sented in Charts 2 and 4. The mean initial level of DNA
synthesis in the 14 tumors of this group is given in Table 1,
together with the mean levels after 2 days of culture, in the
presence and absence of insulin. The mean initial level was
to
the
mean
2-day
level
in the
insulin-stimulated
explants, indicating that a majority of tumors were of a type
similar to that of Chart 3. For the study of correlations with
the time course of DNA polymerase activity and evaluation of
the effect of various pretreatments, the tumors were individ
ually classified according to their initial level of DNA synthesis.
They were designated as inactive when the initial level was
closer to the 2-day level of the control explants than of the
insulin-treated explants (Chart 2) and as activated when the
reverse was true (Charts 3 and 4). According to this classifica
tion, 5 tumors
were inactive,
whereas
9 were activated
(Table
2).
Variations in the initial level of DNA synthesis were due to
differences
in reactivity of the tumor tissue rather than to
systemic environmental
factors of the host. This was demon
strated in control experiments comparing pairs of tumors from
a same rat and showing that inactive and activated tumors
could coexist in the same rat.
DNA polymerase activity was extremely variable from
tumor to tumor. This variability did not allow us to carry out
useful comparisons
between
group means. Therefore,
in order
to analyze the results, the tumors were classified as inactive or
activated, according to the initial level of DNA polymerase
1971
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61
J. C. Heuson and N. Legros
activity, with the same definition as for DNA synthesis. In 10
of the 12 tumors where valid measurements were obtained,
insulin produced a significant increase of activity after 48 or
72 hr of culture. Three were classified as inactive; the
0
10,000
150
.-
;
E
@-.-
E
E
@
and reached
75
.
@n
a-.
I
In view of the finding that the tumors were either inactive
or activated at onset of culture with respect to both DNA
I
synthesis
@
;
DURATION OF CULTURE (DAYS)
Chart 4. Effect of insulin on explants of another tumor from an
untreated rat. High initial level of DNA synthesis and of DNA poly
merase activity followed by a drop, even in the presence of insulin. For
DNA polymerase assay, the substrate concentration was dNTP X 2.
Other conditions and symbols as in Chart 2.
1PretreatmentDNAsynthesisTable
(mean values)
tissue)Initial
(dpm/mg
cultureWith
2 days of
levelAfter
insulinUntreated
insulin
5782
4728
1779
1441
1360
between
the initial
level of DNA
synthesis in these
groups is highly significant, p < 0.002 (Mann-Whitney U test).
Table 2
No. of inactive and activated tumorsa
DNA synthesis
Inactive
Activated
DNA polymerase activity
Inactive
Activated
Untreated5937Insulin4816Diabetes10b52
and DNA polymerase
were classified as inactive or activated
with regard either to
when the initial level in culture was closer to the 2-day level of
the control explants than of the insulin-treated explants (Chart 2).
They were defined as activated when the reverse was true (Chart 3).
b All tumors from diabetic rats were inactive with regard to DNA
synthesis. This contrasts significantly with the untreated group, where a
majority of tumors were activated (p < 0.005, Fisher's exact
probability test).
62
and since this was
as classified
either on the basis of initial level of DNA
synthesis, 4 and 8 tumors, respectively, or of initial DNA
polymerase activity, 1 and 6 tumors, respectively. All tumors
fell in the same categories by both classifications, except 2
(DNA polymerase :activated ; DNA synthesis:inactive). These
data were similar to those found in the untreated group of
rats. It is concluded that insulin pretreatment did not affect
the parameters under consideration.
Alloxan Diabetes. Tumors were excised for culture 9 to 17
days after induction of alloxan diabetes. Thirteen tumors
regressing during the diabetic period were studied and 10 were
found to be insulin dependent; of the remaining 3, 1 had a
negligible rate of DNA synthesis, and 2 were insulin independ
ent. Three tumors growing despite the diabetic state were also
cultured; all 3 were insulin independent.
Table 1 gives the mean levels of DNA synthesis at onset and
after 2 days of culture, with and without insulin, in the 10
dependent
DNA synthesis or to DNA polymerase activity. They were defined as
activity
thought to reflect the state of activation of their DNA
synthesizing process in vivo, an attempt was made to influence
it by modifying the insulin level in the animal. Therefore,
tumor-bearing rats were subjected either to insulin adminis
tration or to alloxan diabetes prior to excision of the tumors
for culture.
Insulin Administration.
Insulin was administrated
for
periods ranging from 13 to 24 days before tumor excision.
Thirteen such tumors were studied in culture and 12 were
found to be insulin dependent. Table 1 gives their mean levels
of DNA synthesis at onset and after 2 days of culture, with
and without insulin. DNA polymerase activity was measured in
10 tumors, and insulin produced a significant increase in 7.
Table 2 shows the relative number of inactive and activated
tumors
Without
(14 tumors)
Insulin (12 tumors)
5199
Diabetes (10 tumors)5024a 1569a5292
inactive
2 or 3 days. This is
Effect of in Vivo Pretreatment
(U
z
a Tumors
value only after
(U
I
Pretreatment
its lowest
distinctly observed in Chart 4.
V
V5000
a The difference
remaining 7 were classified as activated (Table 2). All tumors
fell in the same categories as for DNA synthesis, except one in
which DNA polymerase was activated and yet DNA synthesis
was inactive. This demonstrates the close parallelism of DNA
synthesis and DNA polymerase activity as influenced by
insulin during culture. However, one major difference was
observed in all tumors of the activated type when daily meas
urements were carried out. In the absence of insulin, DNA
synthesis always dropped close to its minimal value within 24
hr, whereas DNA polymerase activity declined more slowly
tumors.
The mean initial
level of DNA synthesis
was much lower than in the untreated group of rats (p <
0.002). In contrast, after 2 days of culture, the mean insulin
stimulated level was close to the corresponding one in the
untreated group of rats. Table 2 shows that all 10 tumors were
inactive with respect to initial level of DNA synthesis. This is
in sharp contrast to the relative numbers of inactive and
activated tumors in the untreated group of rats (p < 0.005).
These results indicate that, in tumors regressing after induction
CANCER RESEARCH VOL. 31
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DNA Polymerase in Cultures ofMammary
Carcinoma
of diabetes, the DNA-synthesizing process has become inactive
in vivo but still can be fully reactivated
by insulin in vitro.
Measurable activity of DNA polymerase was found in 8 of
the 10 tumors. Insulin significantly enhanced this activity in 7
tumors after 2 or 3 days of culture. Classification with regard
to initial activity yielded 5 inactive and 2 activated tumors
(Table 2). The latter 2 did not fit the classification based on
DNA synthesis. The different time course of the two param
eters is well illustrated in Chart 5.
E
E
V
(U
(F,
4
Lai
I
Insulin-independent Tumors
0
a.
Six tumors
of the present
series were fully insulin independ
4
z
ent (cultures of 3 to 4 days). Two belonged to the untreated
group of rats and I to the insulin-pretreated group; the other 3
were those growing in alloxan-diabetic rats. A typical culture
0
(tumor from an untreated rat) is shown in Chart 6. This cul
ture is representative of all 6 independent tumors, although in
1 the initial level of DNA synthesis and of DNA polymerase
activity was low, with a subsequent rise. It clearly shows that
in such tumors insulin has no effect whatsoever on DNA
synthesis and DNA polymerase activity throughout culture.
Two additional tumors, which were regressing in diabetic
rats were classified as insulin independent according to the
definition used in this paper, since insulin failed to increase
DNA synthesis significantly on Day 2 of culture. However,
insulin produced a small but significant increase of DNA
polymerase activity on Day 2 in 1 and of DNA synthesis on
Day 1 and 3 in the other. It would therefore appear that these
tumors had retained a slight degree of insulin dependence and
should not be called insulin independent without qualification.
In control experiments (not shown), it was found twice that
insulin-dependent and insulin-independent tumors coexisted in
the same rat, indicating that insulin dependence is a property
of the tumor tissue rather than of the host. Moreover, insulin
dependence was totally unrelated to the age of the rats within
the range of 160 to 225 days.
10,000
1,000
1
DISCUSSION
In earlier studies (6, 7) on the carcinogen-induced,
mone-dependent
E
E
a.
JANUARY
con
of the enzyme
from the cytoplasm
into the nucleus.
part in the process
of DNA synthesis
but do not
(1 5). Another
technical
problem
is that of native
versus denatured DNA used as primer. According to Ove and
Laszlo ( 17), native DNA polymerase may be the rate-limiting
enzyme for in vivo DNA synthesis, whereas Iwamura et aL
Chart 5. Effect of insulin on explants of a tumor from a rat made
diabetic 9 days before. Inactive with respect to DNA synthesis;
symbols as in Chart 2.
special
polymerase
1
2
4
DURATION OF CULTURE (DAYS)
activity.
under
0
z
to DNA polymerase
studied
4
0
0.
with respect
polymerase,
settle clearly the respective role of the nuclear and cytoplasmic
enzymes. In our work, the enzyme activity was extracted in a
Ca2@-free medium; it has been shown that this procedure
allows release of an important part of the nuclear DNA
I
activated
The nuclear
important
4
(U
/-%--@%‘.%%‘
hor
of the female Sprague
These studies favor the view that DNA polymerase plays an
500
I@5P0O
carcinoma
Dawley rats in organ culture, it was suggested that stimulation
of DNA synthesis by insulin probably involved activation or
induction of enzyme systems related to this process. If so,
insulin would be expected to bring about parallel changes in
DNA synthesis and enzyme activity. In addition, insulin
should not affect the enzyme activity in tumors which are
insulin independent for DNA synthesis. In the experiments
reported here, where DNA synthesis and DNA polymerase
were measured simultaneously, these expectations were fully
verified.
In the present work, DNA polymerase was assayed in the
supernatant fraction of the tumor tissue homogenate. This
fraction might not include some activity firmly bound in the
location
V
@
mammary
ditions of extraction (5), appears to be more closely associated
with DNA synthesis than is the cytoplasmic activity . In other
studies (4, 12), increase in the nuclear polymerase associated
with stimulated DNA synthesis seems to result from a trans
0@
E
3
Chart 6. Lack of effect of insulin on DNA synthesis and DNA po
lymerase activity in explants of a tumor from an untreated rat. Insulin
independent tumor. Conditions and symbols as in Chart 2.
nucleus.
E
2
DURATION OF CULTURE (DAYS)
Conditions
and
( 10) reach the conclusion that polymerase preferring
denatured DNA would be the active enzyme.
With these limitations in mind, it would appear that the
assay method for DNA polymerase used here is suitable for
1971
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63
J. C. Heuson and N. Legros
studying correlations with DNA synthesis. Thus, stimulation
of DNA synthesis under various conditions in several mam
malian tissues has been accompanied by a concomitant rise in
DNA polymerase activity. This has been reported in regen
erating liver after partial hepatectomy (2, 3, 16), in liver of
diabetic
rats after insulin administration
(21), during lympho
cyte transformation by phytohemagglutinin in vitro (14), and
in mouse mammary tissue stimulated by insulin in organ cul
ture (13, 19). The close correlation between the two processes
suggests that in these systems the soluble DNA polymerase
is a
reliable indicator of DNA synthesis.
In a large majority of the tumors, whether insulin
dependent or independent, DNA synthesis and DNA poly
merase
activity
ran a closely
Our observations
discussed
synthesis
parallel
time course
in the insulin-dependent
in culture.
tumors
will be
first. Because of widely different levels of DNA
at onset of culture, they were classified as inactive or
activated according to their initial levels. In inactive tumors
(Chart 2), insulin, after a lag period of more than 4 hr, induced
a concomitant and parallel rise in DNA synthesis and poly
merase
activity.
This suggests that insulin produces
a delayed
activation or an induction of DNA polymerase and probably
of other enzyme systems that are limiting for DNA synthesis.
It is in agreement with our previous interpretation that insulin
stimulation of DNA synthesis is not merely a permissive effect
which, by making energy and substrates available, would
operate on mechanisms already fully equipped and ready to
function.
An interesting observation was the finding that in a majority
of the tumors, both from untreated and from insulin-pre
treated rats, DNA synthesis and DNA polymerase activity were
fully activated at onset of culture and could not be further
stimulated by insulin during culture (Chart 3). In some tumors
(Chart 4), these two values even dropped in the presence of
insulin. Nonetheless, these tumors were insulin dependent
inasmuch as insulin created a large and significant difference in
both DNA synthesis and DNA polymerase activity during
culture.
Whereas in most activated tumors DNA synthesis and DNA
polymerase activity were both at a high initial level, it occur
red in occasional tumors, especially from diabetic rats, that
only DNA polymerase activity was high at onset of culture,
while DNA synthesis was low and required insulin for activa
tion in vitro (Chart 5). It is obvious that, in such cases, DNA
polymerase was no longer rate limiting for DNA synthesis. The
same holds true in the control explants of typical activated
tumors, when inactivation takes place as a result of lack of
insulin: DNA synthesis drops more abruptly than DNA polym
erase activity. A comparable divergence has been described
during certain phases of liver regeneration in vivo (3, 16) and
in insulin-stimulated mammary tissue after culture periods
exceeding 72 hr (13). It is clear that DNA synthesis may
return to low rates in the presence of fully activated DNA
polymerase. In the case of the tumor of Chart 5, cessation of
insulin stimulation in the diabetic animal may account for the
particular features observed in culture.
The initial level of the DNA synthesizing process (rate of
thymidine-3 H incorporation and DNA polymerase activity) in
culture was assumed to reflect the state of the tumor in vivo at
64
the time of its excision. The existence of tumors both inactive
and activated with respect to this process raises the question of
the regulatory mechanism involved in vivo and of its specif
icity. A partial answer was given by the observation that all
tumors regressing in diabetic rats were inactive with respect to
DNA synthesis, although their ability to become activated in
culture under the effect of insulin remained intact. This sug
gests that insulin may play the same part in the in vivo regula
tion of this process as in vitro. This interpretation is supported
by experimental results reported elsewhere showing that
alloxan diabetes produced a rapid regression of most tumors,
comparable to what is seen after oophorectomy. Moreover,
tumors growing despite the diabetic state (8 such tumors were
studied) were precisely insulin independent or hardly sensitive
to insulin in vitro. Conversely, administration of insulin in rats
resulted in a dramatic (8.3-fold) acceleration of tumor growth
(8). That this effect of insulin was not mediated through an
increase in pituitary growth hormone secretion was recently
demonstrated in hypophysectomized
rats receiving prolactin.
Insulin significantly stimulated growth on the regressed tumors
under these circumstances (J. C. Heuson and N. Legros,
unpublished results).
While variations of DNA synthesis could be induced in vivo
by changes in environmental factors such as the level of
insulin, it was also shown that inactive and activated tumors
coexisted in the same rat. This seems to indicate a difference
between tumors in their response to a given environment.
Since the normal mammary tissue has a low initial level of
DNA synthesis and of DNA polymerase activity (1 3) and,
since this was true also in a spontaneous virus-associated mam
mary adenocarcinoma
of the mouse (19), it would appear that
the state of activation exhibited here by a majority of the rat
tumors is a distinctive characteristic of these particular tumors,
which seem to be overresponsive to stimulating factors,
possibly insulin, in vivo. The tumor of Chart 4 might represent
an extreme example of such hypersensitivity. This property of
the rat tumor is strikingly reminiscent of the work of Pitot and
Morris ( 18) on minimal deviation hepatomas. In these tumors,
tyrosine transaminase is overresponsive to induction by
cortisone, and the high stimulated level that exists in the intact
animal is reverted back to almost normal after adrenalectomy.
This abornmally high sensitivity to stimulating factors may
tentatively be interpreted as a result of derepression and
escape from inhibitory control mechanisms. Insulin depend
ence might then represent
a further
step in the same direction.
Insulin-independent
tumors, which cannot be distinguished
from the dependent ones on histological grounds, were shown
previously to exhibit a high mitotic activity unaffected by
insulin in culture (6). In the present work, DNA polymerase
was found to be equally unaffected by insulin, and its activity
was closely parallel to that of DNA synthesis. Insulin indepen
dence is also a property of the immature normal mammary
gland (20).
The loss of insulin dependence, as observed in some of the
7 ,12-dimethylbenz(a)anthracene-induced
mammary carcino
mas of the Sprague-Dawley rats, appears to accompany
the specific ability to grow during diabetes. Turkington and
Hilf (19) recently reported a study on a transplantable, rapidly
proliferating mammary carcinoma of the Fischer rat. This
CANCER RESEARCH VOL. 31
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DNA Polymerase in Cultures ofMammary
tumor was independent of insulin for DNA synthesis in organ
culture. It should be of interest to determine whether such
tumor is transplantable and able to grow in diabetic rats.
Carcinoma
9. Huggins, C., Grand, L. C., and Brillantes, F. P. Mammary Cancer
Induced by a Single Feeding of Polynuclear Hydrocarbons, and Its
Suppression.Nature, 189: 204—207,1961.
10. Iwamura, Y., Ono, T., and Morris, H. P. The Heterogeneity of DNA
Polymerases in Rat Liver and Hepatomas. Cancer Res., 28:
2466—2476,1968.
ACKNOWLEDGMENTS
We are indebted to Dr. J. Dumont and to Dr. H. J. Tagnon for
valuable discussions and for their help in the preparation of the manu
script.
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JANUARY
1971
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65
Effect of Insulin on DNA Synthesis and DNA Polymerase
Activity in Organ Culture of Rat Mammary Carcinoma, and the
Influence of Insulin Pretreatment and of Alloxan Diabetes
J. C. Heuson and N. Legros
Cancer Res 1971;31:59-65.
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