[CANCER RESEARCH 31, 59—65, January 1971]

hormone-dependent mammary carcinomas of the rat in organculture. It was also shown that this effect was not mediatedthrough an increase in glucose uptake and utilization by theexplants and, therefore, appeared not to be a simple permissiveeffect bearing on energy-yielding reactions. Instead, insulinseemed likely to act on processes more directly related to thecontrol of DNA synthesis. Another interesting feature wasthat, in addition to tumors with insulin-dependent cell proliferation, others displayed an intense mitotic activity occurringspontaneously and independently of the presence of insulin invitro. These two types of tumors appeared to be identical onhistological examination. The loss of insulin dependence in thesecond type was thought to result from alteration of thenormal growth-regulating mechanisms inherent in the neoplastic process.

The contention that insulin seemed to act on processesrelated to the control of DNA synthesis led us to study itseffect on DNA polymerase activity in the tumor explants. Thisenzyme system was selected because, as reported by Lockwood et aL (13), initiation of DNA synthesis by insulin in thenormal epithelial cells of mammary explants from mature miceinvolved emergence of DNA polymerase activity in these cells.The same relationship between DNA synthesis and activity ofthis enzyme was observed by other authors in various mammalian tissues subjected to the proper stimulus (2, 3, 14, 16,21).

The present work studies the effect of insulin on DNAsynthesis and DNA polymerase activity in explants of bothinsulin-dependent and insulin-independent rat mammarytumors. It also examines the effect of insulin pretreatment andof alloxan diabetes in vivo on the properties of the tumors inculture. The purpose of these studies was to investigate themechanism of the growth-promoting effect of insulin on therat mammary tumor in vitro and to derive conclusions on thepossible role of insulin on the control of their growth in vivo.

MATERIALS AND METHODS

Animals. Mammary tumors were induced in Sprague-Dawleyfemale rats by a single feeding of 7,l2-dimethylbenz(a)-anthracene at age 50 days (9). Tumors studied in culture wereobtained both from untreated rats and from rats receiving oneof the following treatments. One group of rats received 2.5 i.u.

JANUARY 1971 59

Effect of Insulin on DNA Synthesis and DNA PolymeraseActivity in Organ Culture of Rat Mammary Carcinoma,and the Influence of Insulin Pretreatment and of AlloxanDiabetes1

J. C. HeusonandN. LegrosDepartment of Internal Medicine2 and Laboratory of Clinical Investigation, Institut Jules Bordet, Centre des Tumeurs de l'UniversitéLibre deBruxelles, Brussels,Belgium

SUMMARY

As previously reported , 7,1 2-dimethylbenz(a)anthraceneinduced mammary carcinomas of the rat are usually insulindependent for cell proliferation in organ culture, although afew proliferate readily in the complete absence of insulin. Thisproblem was further investigated by studying the effect ofinsulin on DNA polymerase in the cultured explants.

It was found for most tumors that DNA synthesis and DNApolymerase activity ran a closely parallel time course. Stimulation of DNA synthesis was delayed in onset and was accompanied by a concomitant rise in DNA polymerase activity. Intumors with insulin-independent DNA synthesis, DNA polymerase was also insulin independent. These observations supportour earlier interpretation that the stimulating propertyof insulin is not merely a permissive effect, mediated throughenergy-yielding reactions, but rather involves activation orinduction of enzyme systems responsible for DNA synthesis.

The level of the DNA-synthesizing process at onset ofculture varied markedly from tumor to tumor, even in thesame rat. It ranged from low to highly activated. The activatedstate, which was most frequently encountered, would seem toresult from an “overresponsiveness―of the process to stimulating factors in vivo. The fact that inactivation invariablyoccurred after induction of alloxan diabetes, together withother lines of evidence, suggest that insulin may be one ofthese factors.

Hypersensitivity to stimulating factors and insulin independence possibly represent successive steps toward escape fromthe normal growth-regulating mechanisms. Loss of insulindependence, as revealed in culture, appears to accompany thespecific ability of the tumor to grow in diabetic rats.

INTRODUCTION

In earlier studies (6, 7), it was demonstrated that insulinstimulated cell proliferation in explants of carcinogen-induced,

I This work was supported in part by Contract EURATOM

ULB-PISE 026-634-BIAC and by a grant of F&lérationBelgo-Luxembourg eoise des Industries du Tabac to the EuropeanOrganization for Researchon Treatment of Cancer.

@ This department is affiliated with the European Organization forResearch on Treatment of Cancer.

Received May 11, 1970; accepted September 23, 1970.

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J. C. Heuson and N. Legros

(1 .zCi) of dATP-3 H (specific activity, 4.8 to 15.1Ci/mmole; Schwarz BioResearch, Inc., Orangeburg N. Y.);100 pg of heat-denatured salmon sperm DNA; and 0.05 ml ofenzyme preparation (supernatant traction). However, the basicconcentration of the 4 dNTP's (dNTP X 1) was increased inthe reaction mixture, for reasons explained below, usually by afactor of 6 (dNTP X 6) or, when enzyme activity was low, bya 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% caseinsolution containing 333 mj.zmoles of ATP and 1.25 ml of coldN PCA. The procedure described by Lockwood et aL (13) forwashing the acid precipitate was not used here because it gavehigh radioactivity values in the blanks. It was replaced by aprocedure based on a method described by Younger et aL(21). The precipitate was centrifuged at 2000 rpm for 5 min inthe cold. The sediment was dissolved at 0°in 0.5 ml of 0.5 NNaOH. The resulting solution was completed by adding, inrapid succession@0.5 ml of water containing 666 mpmoles ofATP and 1 ml of N PCA. The procedure was repeated 3 times.It yielded very low blanks and a high recovery of theheat-denatured DNA. The final precipitate was washed twicewith ethanol:ether (3: 1), dissolved in Hyammne, and countedin a liquid scintillation spectrometer. The results werecorrected for quenching and expressed as jipmoles of dATP ordpm/mg of tissue, wet weight, in the enzyme preparation.

The reason for increasing 2- to 6-fold the concentration ofthe 4 dNTP's in the reaction mixture over that used by Lockwood et aL ( 13) for the mouse mammary tissue is shown inChart 1. The substrate concentration dNTP X I yielded, forthis tumor-enzyme preparation, a reaction velocity belowVmax and nonlinear kinetics during incubation periods of 60mm. Concentrations3 or 6 timeshigher(dNTPX 3 or X 6)gave maximal velocity and linear kinetics.

Omission of the dNTP's from the reaction mixture produced a decrease in dATP-3 H incorporation. The decrease was20% when dCTP was omitted, 50% when dGTP or TTP wasomitted, and 80% when all three were omitted. This showsthat the activity is mainly composed of the replicative enzyme(11).

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of Novo-Lente insulin (Novo A/S, Copenhagen, Denmark)per 100 g of body weight s.c. daily, except on Sundays, forvarying periods of time before tumor excision. In addition,they were given a 10% glucose solution as drinking fluid inorder to protect them from lethal hypoglycemia. Anothergroup was made diabetic by a single i.p. injection of alloxan(12 mg/lOO g of body weight). For reduction of mortality inthe latter group, insulin replacement therapy was started onthe following day and given in decreasing doses for severaldays. The day following cessation of insulin administrationwas taken as Day 0 of diabetes. Rats were considered diabeticwhen the blood serum glucose was in excess of 350 mg/lOO ml(glucose oxidase method, glucostat reagents, WorthingtonBiochemical Corp., Freehold, N. J.).

Culture Method. Explants of freshly excised tumor tissuewere prepared for organ culture by placing 12 fragments ofabout 1 cu mm on a Millipore filter disc. Each disc was maintamed at the surface of 2 ml of the chemically definedMedium 199 enriched with glucose (0.25 g/l00 ml) for cultureperiods of 2 to 4 days at 37°.The gas phase was 95% 02 and5% CO2 . The medium was changed daily and contained bovinecrystalline insulin (24.3 i.u./mg, Calbiochem, Los Angeles,Calif.) at a concentration of 40 j.zg/ml. Control explants werecultured in parallel without insulin.

Assay for DNA Synthesis and DNA Polymerase Activity.DNA synthesis and DNA polymerase activity were measuredsimultaneously in the explants. The term explant is used hereas a unit to designate a group of 12 fragments placed on aMillipore filter disc. The determinations were carried out on 3to 4 replicate explants.

DNA synthesis was determined after a 4-hr exposure to 0.5j.zCi/ml of thymidine-3H (specific activity, 1.15 Ci/mmole,Calbiochem, Los Angeles, Calif.). The explants were washed 4times in Earle's base containing 0.1% glucose and mMthymidine, blotted, weighed, homogenized at 0°in 0.5 ml of asolution 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% ofthe acid-insoluble radioactivity was resuspended in 0.5 ml ofmM thymidine. The acid-insoluble material was precipitatedby adding 1 ml of N PCA.3 The precipitate was washed twicewith 2 ml of N PCA and twice with ethanol:ether (3: 1). It wasdissolved in 1 ml of Hyamine and counted in a liquidscintillation spectrometer. The results were corrected forquenching and expressed as dpm/mg of tissue, wet weight.

DNA polymerase activity was estimated by a modificationof the methods described by Bollum (1) and Lockwood et aL(13). The supernatant fluid of the 800 X g centrifugation wasrecentrifuged in the cold at 90,000 X g for 60 mm or at200,000 X g for 30 mm. The supernatant fluid was used as theenzyme preparation.

The basic reaction mixture for assay of DNA polymeraseactivity contained in 0.25 ml: 20 j.tmoles of Tris-HC1 buffer(pH 7.4); 2 jimoles of MgC12; 0.25 @.tmoleof 2-mercaptoethanol; 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

3The abbreviations used are: PCA, perchloric acid; dNTP, deoxynucleoside triphosphate.

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DNA Polymerase in Cultures ofMammary Carcinoma

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Chart 2. Effect of insulin on DNA synthesis and on DNA polymeraseactivity (substrate concentration, dNTP X 6) in explants of a tumorfrom an untreated rat. Low initial level of the 2 parameters (inactivetumor). DNA synthesis expressed as dpm of thymidine-3 H incorporatedinto cell DNA(4-hr exposure)/mg of tissue. Results of DNApolymeraseassay expressed as dpm of dATP-3 H incorporated into primer DNA(60-mm incubation)/mg tissue in the enzyme preparation. DNAsynthesis 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, @--

RESULTS

The effect of insulin on DNA synthesis and on DNA polymerase activity in culture of 2 to 4 days was studied in a seriesof 45 tumors. Sixteen of these were obtained from untreatedrats, 13 were from insulin-pretreated rats, and 16 were fromalloxan-diabetic rats. It was found that a majority of thetumors (36 tumors) needed insulin for DNA synthesis, whereas8 were insulin independent and 1 showed a negligible rate ofDNA synthesis. Since the stimulating effect of insulin wasdelayed in onset (cf. below), tumors were defined as insulindependent when insulin produced a significant increase inDNA synthesis on Day 2 of culture.

Insulin-dependent Tumors

Untreated Rats. Fourteen of the I 6 tumors in this groupwere insulin dependent. Chart 2 demonstrates the effect ofinsulin on such a tumor during culture. DNA synthesis andDNA polymerase activity ran a closely parallel time course.Their values were low at onset of culture and were unaffectedby insulin during the first 4 hr. Thereafter, they rose sharply inthe presence of insulin, while they remained low in its absence.

The lack of effect of insulin during the first 4 hr of culturewas a constant finding in all 12 insulin-dependent tumorswhere this particular point was investigated. In view of thisobserved constancy, the initial measurements were carried out,in the other tumors, only on the control explants.

The insulin dependence expressed itself in several differentways, illustrated in the following charts.

Chart 3 shows the result of a culture experiment performedwith another insulin-dependent tumor. The results differ fromthose of Chart 2 in that the initial values of DNA synthesis andof DNA polymerase activity were high. The effect of insulinwas only to maintain these high values throughout culture. Inthe absence of insulin, they dropped abruptly. In some

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Chart 3. Effect of insulin on explants of another tumor from anuntreated rat. High initial level of DNA synthesis and of DNA polymerase activity (activated tumor). Conditions and symbols as in Chart 2.

tumors, daily measurements disclosed an irregular time courseof DNA synthesis in the insulin-treated explants, suggestingthat partial synchronization of the mitotic activity may haveoccurred.

Chart 4 illustrates the behavior of still another type ofinsulin-dependent tumor; 2 cases of this type were found inthe group of untreated rats. DNA synthesis and DNA polymerase activity had high initial values, followed by a sharpdrop both in the presence and absence of insulin. In thistumor, the time course of DNA polymerase activity displayedparticular features that will be described below.

Thus, the level of DNA synthesis and of DNA polymeraseactivity at onset of culture, which may be assumed to reflectthe state of the tumor in vivo at the time of excision forculture, varied among tumors between the extremes represented in Charts 2 and 4. The mean initial level of DNAsynthesis in the 14 tumors of this group is given in Table 1,together with the mean levels after 2 days of culture, in thepresence and absence of insulin. The mean initial level wasclose to the mean 2-day level in the insulin-stimulatedexplants, indicating that a majority of tumors were of a typesimilar to that of Chart 3. For the study of correlations withthe time course of DNA polymerase activity and evaluation ofthe effect of various pretreatments, the tumors were individually classified according to their initial level of DNA synthesis.They were designated as inactive when the initial level wascloser to the 2-day level of the control explants than of theinsulin-treated explants (Chart 2) and as activated when thereverse was true (Charts 3 and 4). According to this classification, 5 tumors were inactive, whereas 9 were activated (Table2).

Variations in the initial level of DNA synthesis were due todifferences in reactivity of the tumor tissue rather than tosystemic environmental factors of the host. This was demonstrated in control experiments comparing pairs of tumors froma same rat and showing that inactive and activated tumorscould coexist in the same rat.

DNA polymerase activity was extremely variable fromtumor to tumor. This variability did not allow us to carry outuseful comparisons between group means. Therefore, in orderto analyze the results, the tumors were classified as inactive oractivated, according to the initial level of DNA polymerase

JANUARY 1971 61

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Table1PretreatmentDNAsynthesis

(mean values)(dpm/mgtissue)Initial

levelAfter

2 days ofcultureWith

insulin WithoutinsulinUntreated

(14 tumors)Insulin (12 tumors)Diabetes (10 tumors)5024a

51991569a5292

17795782 14414728 1360

Untreated5937Insulin4816Diabetes10b52

J. C. Heuson and N. Legros

activity, with the same definition as for DNA synthesis. In 10of the 12 tumors where valid measurements were obtained,insulin produced a significant increase of activity after 48 or72 hr of culture. Three were classified as inactive; the

remaining 7 were classified as activated (Table 2). All tumorsfell in the same categories as for DNA synthesis, except one inwhich DNA polymerase was activated and yet DNA synthesiswas inactive. This demonstrates the close parallelism of DNAsynthesis and DNA polymerase activity as influenced byinsulin during culture. However, one major difference wasobserved in all tumors of the activated type when daily measurements were carried out. In the absence of insulin, DNAsynthesis always dropped close to its minimal value within 24hr, whereas DNA polymerase activity declined more slowlyand reached its lowest value only after 2 or 3 days. This isdistinctly observed in Chart 4.

Effect of in Vivo Pretreatment

In view of the finding that the tumors were either inactiveor activated at onset of culture with respect to both DNAsynthesis and DNA polymerase activity and since this wasthought to reflect the state of activation of their DNAsynthesizing process in vivo, an attempt was made to influenceit by modifying the insulin level in the animal. Therefore,tumor-bearing rats were subjected either to insulin administration or to alloxan diabetes prior to excision of the tumorsfor culture.

Insulin Administration. Insulin was administrated forperiods ranging from 13 to 24 days before tumor excision.Thirteen such tumors were studied in culture and 12 werefound to be insulin dependent. Table 1 gives their mean levelsof DNA synthesis at onset and after 2 days of culture, withand without insulin. DNA polymerase activity was measured in10 tumors, and insulin produced a significant increase in 7.Table 2 shows the relative number of inactive and activatedtumors as classified either on the basis of initial level of DNAsynthesis, 4 and 8 tumors, respectively, or of initial DNApolymerase activity, 1 and 6 tumors, respectively. All tumorsfell in the same categories by both classifications, except 2(DNA polymerase :activated ; DNA synthesis:inactive). Thesedata were similar to those found in the untreated group ofrats. It is concluded that insulin pretreatment did not affectthe parameters under consideration.

Alloxan Diabetes. Tumors were excised for culture 9 to 17days after induction of alloxan diabetes. Thirteen tumorsregressing during the diabetic period were studied and 10 werefound to be insulin dependent; of the remaining 3, 1 had anegligible rate of DNA synthesis, and 2 were insulin independent. Three tumors growing despite the diabetic state were alsocultured; all 3 were insulin independent.

Table 1 gives the mean levels of DNA synthesis at onset andafter 2 days of culture, with and without insulin, in the 10dependent tumors. The mean initial level of DNA synthesiswas much lower than in the untreated group of rats (p <0.002). In contrast, after 2 days of culture, the mean insulinstimulated level was close to the corresponding one in theuntreated group of rats. Table 2 shows that all 10 tumors wereinactive with respect to initial level of DNA synthesis. This isin sharp contrast to the relative numbers of inactive andactivated tumors in the untreated group of rats (p < 0.005).These results indicate that, in tumors regressing after induction

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a The difference between the initial level of DNA synthesis in thesegroups is highly significant, p < 0.002 (Mann-Whitney U test).

Table 2

No. of inactive and activated tumorsa

DNA synthesis DNA polymerase activity

Pretreatment Inactive Activated Inactive Activated

a Tumors were classified as inactive or activated with regard either toDNA synthesis or to DNA polymerase activity. They were defined asinactive when the initial level in culture was closer to the 2-day level ofthe 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 DNAsynthesis. This contrasts significantly with the untreated group, where amajority of tumors were activated (p < 0.005, Fisher's exactprobability test).

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of diabetes, the DNA-synthesizing process has become inactivein vivo but still can be fully reactivated by insulin in vitro.

Measurable activity of DNA polymerase was found in 8 ofthe 10 tumors. Insulin significantly enhanced this activity in 7tumors after 2 or 3 days of culture. Classification with regardto initial activity yielded 5 inactive and 2 activated tumors(Table 2). The latter 2 did not fit the classification based onDNA synthesis. The different time course of the two parameters is well illustrated in Chart 5.

Insulin-independent Tumors

Six tumors of the present series were fully insulin independent (cultures of 3 to 4 days). Two belonged to the untreatedgroup of rats and I to the insulin-pretreated group; the other 3were those growing in alloxan-diabetic rats. A typical culture(tumor from an untreated rat) is shown in Chart 6. This culture is representative of all 6 independent tumors, although in1 the initial level of DNA synthesis and of DNA polymeraseactivity was low, with a subsequent rise. It clearly shows thatin such tumors insulin has no effect whatsoever on DNAsynthesis and DNA polymerase activity throughout culture.

Two additional tumors, which were regressing in diabeticrats were classified as insulin independent according to thedefinition used in this paper, since insulin failed to increaseDNA synthesis significantly on Day 2 of culture. However,insulin produced a small but significant increase of DNApolymerase activity on Day 2 in 1 and of DNA synthesis onDay 1 and 3 in the other. It would therefore appear that thesetumors had retained a slight degree of insulin dependence andshould not be called insulin independent without qualification.

In control experiments (not shown), it was found twice thatinsulin-dependent and insulin-independent tumors coexisted inthe same rat, indicating that insulin dependence is a propertyof the tumor tissue rather than of the host. Moreover, insulindependence was totally unrelated to the age of the rats withinthe range of 160 to 225 days.

1 2 4DURATION OF CULTURE (DAYS)

Chart 5. Effect of insulin on explants of a tumor from a rat madediabetic 9 days before. Inactive with respect to DNA synthesis;activated with respect to DNA polymerase activity. Conditions andsymbols as in Chart 2.

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DISCUSSION

In earlier studies (6, 7) on the carcinogen-induced, hormone-dependent mammary carcinoma of the female SpragueDawley rats in organ culture, it was suggested that stimulationof DNA synthesis by insulin probably involved activation orinduction of enzyme systems related to this process. If so,insulin would be expected to bring about parallel changes inDNA synthesis and enzyme activity. In addition, insulinshould not affect the enzyme activity in tumors which areinsulin independent for DNA synthesis. In the experimentsreported here, where DNA synthesis and DNA polymerasewere measured simultaneously, these expectations were fullyverified.

In the present work, DNA polymerase was assayed in thesupernatant fraction of the tumor tissue homogenate. Thisfraction might not include some activity firmly bound in thenucleus. The nuclear polymerase, studied under special conditions of extraction (5), appears to be more closely associatedwith DNA synthesis than is the cytoplasmic activity . In otherstudies (4, 12), increase in the nuclear polymerase associatedwith stimulated DNA synthesis seems to result from a translocation of the enzyme from the cytoplasm into the nucleus.These studies favor the view that DNA polymerase plays animportant part in the process of DNA synthesis but do notsettle clearly the respective role of the nuclear and cytoplasmicenzymes. In our work, the enzyme activity was extracted in aCa2@-free medium; it has been shown that this procedureallows release of an important part of the nuclear DNApolymerase (1 5). Another technical problem is that of nativeversus denatured DNA used as primer. According to Ove andLaszlo ( 17), native DNA polymerase may be the rate-limitingenzyme for in vivo DNA synthesis, whereas Iwamura et aL( 10) reach the conclusion that polymerase preferringdenatured DNA would be the active enzyme.

With these limitations in mind, it would appear that theassay method for DNA polymerase used here is suitable for

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J. C. Heuson and N. Legros

studying correlations with DNA synthesis. Thus, stimulationof DNA synthesis under various conditions in several mammalian tissues has been accompanied by a concomitant rise inDNA polymerase activity. This has been reported in regenerating liver after partial hepatectomy (2, 3, 16), in liver ofdiabetic rats after insulin administration (21), during lymphocyte transformation by phytohemagglutinin in vitro (14), andin mouse mammary tissue stimulated by insulin in organ culture (13, 19). The close correlation between the two processessuggests that in these systems the soluble DNA polymerase is areliable indicator of DNA synthesis.

In a large majority of the tumors, whether insulindependent or independent, DNA synthesis and DNA polymerase activity ran a closely parallel time course in culture.Our observations in the insulin-dependent tumors will bediscussed first. Because of widely different levels of DNAsynthesis at onset of culture, they were classified as inactive oractivated according to their initial levels. In inactive tumors(Chart 2), insulin, after a lag period of more than 4 hr, induceda concomitant and parallel rise in DNA synthesis and polymerase activity. This suggests that insulin produces a delayedactivation or an induction of DNA polymerase and probablyof other enzyme systems that are limiting for DNA synthesis.It is in agreement with our previous interpretation that insulinstimulation of DNA synthesis is not merely a permissive effectwhich, by making energy and substrates available, wouldoperate on mechanisms already fully equipped and ready tofunction.

An interesting observation was the finding that in a majorityof the tumors, both from untreated and from insulin-pretreated rats, DNA synthesis and DNA polymerase activity werefully activated at onset of culture and could not be furtherstimulated by insulin during culture (Chart 3). In some tumors(Chart 4), these two values even dropped in the presence ofinsulin. Nonetheless, these tumors were insulin dependentinasmuch as insulin created a large and significant difference inboth DNA synthesis and DNA polymerase activity duringculture.

Whereas in most activated tumors DNA synthesis and DNApolymerase activity were both at a high initial level, it occurred in occasional tumors, especially from diabetic rats, thatonly DNA polymerase activity was high at onset of culture,while DNA synthesis was low and required insulin for activation in vitro (Chart 5). It is obvious that, in such cases, DNApolymerase was no longer rate limiting for DNA synthesis. Thesame holds true in the control explants of typical activatedtumors, when inactivation takes place as a result of lack ofinsulin: DNA synthesis drops more abruptly than DNA polymerase activity. A comparable divergence has been describedduring certain phases of liver regeneration in vivo (3, 16) andin insulin-stimulated mammary tissue after culture periodsexceeding 72 hr (13). It is clear that DNA synthesis mayreturn to low rates in the presence of fully activated DNApolymerase. In the case of the tumor of Chart 5, cessation ofinsulin stimulation in the diabetic animal may account for theparticular features observed in culture.

The initial level of the DNA synthesizing process (rate ofthymidine-3 H incorporation and DNA polymerase activity) inculture was assumed to reflect the state of the tumor in vivo at

the time of its excision. The existence of tumors both inactiveand activated with respect to this process raises the question ofthe regulatory mechanism involved in vivo and of its specificity. A partial answer was given by the observation that alltumors regressing in diabetic rats were inactive with respect toDNA synthesis, although their ability to become activated inculture under the effect of insulin remained intact. This suggests that insulin may play the same part in the in vivo regulation of this process as in vitro. This interpretation is supportedby experimental results reported elsewhere showing thatalloxan 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 werestudied) were precisely insulin independent or hardly sensitiveto insulin in vitro. Conversely, administration of insulin in ratsresulted in a dramatic (8.3-fold) acceleration of tumor growth(8). That this effect of insulin was not mediated through anincrease in pituitary growth hormone secretion was recentlydemonstrated in hypophysectomized rats receiving prolactin.Insulin significantly stimulated growth on the regressed tumorsunder these circumstances (J. C. Heuson and N. Legros,unpublished results).

While variations of DNA synthesis could be induced in vivoby changes in environmental factors such as the level ofinsulin, it was also shown that inactive and activated tumorscoexisted in the same rat. This seems to indicate a differencebetween tumors in their response to a given environment.Since the normal mammary tissue has a low initial level ofDNA synthesis and of DNA polymerase activity (1 3) and,since this was true also in a spontaneous virus-associated mammary adenocarcinoma of the mouse (19), it would appear thatthe state of activation exhibited here by a majority of the rattumors 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 representan extreme example of such hypersensitivity. This property ofthe rat tumor is strikingly reminiscent of the work of Pitot andMorris ( 18) on minimal deviation hepatomas. In these tumors,tyrosine transaminase is overresponsive to induction bycortisone, and the high stimulated level that exists in the intactanimal is reverted back to almost normal after adrenalectomy.

This abornmally high sensitivity to stimulating factors maytentatively be interpreted as a result of derepression andescape from inhibitory control mechanisms. Insulin dependence might then represent a further step in the same direction.Insulin-independent tumors, which cannot be distinguishedfrom the dependent ones on histological grounds, were shownpreviously to exhibit a high mitotic activity unaffected byinsulin in culture (6). In the present work, DNA polymerasewas found to be equally unaffected by insulin, and its activitywas closely parallel to that of DNA synthesis. Insulin independence is also a property of the immature normal mammarygland (20).

The loss of insulin dependence, as observed in some of the7 ,12-dimethylbenz(a)anthracene-induced mammary carcinomas of the Sprague-Dawley rats, appears to accompanythe specific ability to grow during diabetes. Turkington andHilf (19) recently reported a study on a transplantable, rapidlyproliferating mammary carcinoma of the Fischer rat. This

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9. Huggins, C., Grand, L. C., and Brillantes, F. P. Mammary CancerInduced by a Single Feeding of Polynuclear Hydrocarbons, and ItsSuppression.Nature, 189: 204—207,1961.

10. Iwamura, Y., Ono, T., and Morris, H. P. The Heterogeneity of DNAPolymerases in Rat Liver and Hepatomas. Cancer Res., 28:2466—2476,1968.

11. Keir, H. M. DNA Polymerases from Mammalian Cells. Prog. NucleicAcid Res. Mol. Biol., 4: 81—128,1965.

12. Littlefield, J. W., McGovern, A. P., and Margeson, K. B. Changes inthe Distribution of Polymerase Activity during DNA Synthesis inMouse Fibroblasts. Proc. Nail. Acad. Sci. U. S., 49: 102—107,1963.

13. Lockwood, D. M., Voytovich, A. E., Stockdale, F. E., and Topper,Y. J. Insulin-dependent DNA Polymerase and DNA Synthesis inMammary Epitheial Cells in Vitro. Proc. Nail. Acad. Sci. U. S., 58:658—664,1967.

14. Loeb, L. A., Agarwal, S. S. and Woodside, A. M. Induction of DNAPolymerase in Human Lymphocytes by Phytohemagglutinin. Proc.Nail. Acad. Sci. U. S., 61: 827—834,1968.

15. Main, R. K., and Cole, L. J. DNA Polymerase in Thymus NuclearFractions Isolated with Ca*. Nature, 203: 646—648, 1964.

16. Ove, P., Jenkins, M. D., and Laszlo, J. DNA Replication andDegradation in MammalianTissue. I. Changes in DNA Polymeraseand Nuclease during Rat Liver Regeneration. Biochim. Biophys.Acta, 174: 629—635,1969.

17. Ove, P., and Laszlo, J. DNA Replication and Degradation in Mammalian Tissues. IV. DNA Synthesis, DNA Polymerase and DNase inNormal Rat Tissues.Oncology,24: 18—20,1969.

18. Pitot, H. C., and Morris, H. P. Metabolic Adaptations in RatHepatomas. II. Tryptophan Pyrrolase and Tyrosine a- KetoglutarateTransaminase.CancerRes.,21:1009—1014,1961.

19. Turkington, R. W., and Hill, R. Hormonal Dependence of DNASynthesis in Mammary Carcinoma Cells in Vitro. Science, 160:1457—1459,1968.

20. Voytovich, A. E., and Topper, Y. J. Hormone-dependent Differentiation of Immature Mouse Mammary Gland in Vitro. Science,158: 1326—1327,1967.

21. Younger, L. R., King, J., and Steiner, D. F. Hepatic ProliferativeResponse to Insulin in Severe Alloxan Diabetes. Cancer Res., 26:1408—1414,1966.

tumor was independent of insulin for DNA synthesis in organculture. It should be of interest to determine whether suchtumor is transplantable and able to grow in diabetic rats.

ACKNOWLEDGMENTS

We are indebted to Dr. J. Dumont and to Dr. H. J. Tagnon forvaluable discussions and for their help in the preparation of the manuscript.

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JANUARY 1971 65

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1971;31:59-65. Cancer Res   J. C. Heuson and N. Legros  Influence of Insulin Pretreatment and of Alloxan DiabetesActivity in Organ Culture of Rat Mammary Carcinoma, and the Effect of Insulin on DNA Synthesis and DNA Polymerase

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