[CANCER RESEARCH 40, 3158-31 62, September 1980]0008-5472/80/0040-0000$02.O0
Comparative Effects of Estrogen and Antiestrogens on Enzyme Activities inR3230AC Rat Mammary Tumors and Uteri of Tumor-bearing Animal&
Ellen A. Rorke and Benita S. Katzenellenbogen2
Department of Physiology and Biophysics, University of Illinois, and School of Basic Medical Sciences, University of Illinois College of Medicine, Urbana,Illinois61801
ABSTRACT
Growth of the R3230AC mammary tumor, an ovarian-autonomous but estrogen-responsive tumor in Fischer 344 rats, ismarkedly depressed by administration of antiestrogen or highdoses of estradiol. We have examined the effects of antiestrogens and estradiol on the activity of several enzymes known tobe modulated by estrogen, namely, glucose-6.-phosphate dehydrogenase (G6PD), NADP-dependent malic dehydrogenase(malic enzyme), a-glycerol phosphate dehydrogenase, and peroxidase. In addition, we have compared the effects of antiestrogens and estradiol on these enzymatic activities in mammarytumors and uteri of R3230AC tumor-bearing animals to enablea comparison of hormonal and antihormonal action in thesetwo estrogen-sensitive tissues. Rats were treated with antiestrogen or estradiol for 24 days beginning on the day of tumortransplantation.
In mammary tumors, estradiol (25 @tgs.c. in 0.1 5 M NaCI perday per rat) increased the activity of G6PD and malic enzyme2-fold (expressed on a tissue weight or mg protein basis) anddecreased the activity of a-glycerol phosphate dehydrogenase2-fold; ovariectomy and antiestrogen treatments (250 @sgs.c.in 0. 15 M NaCI per day of 1-{24p-(3,4-dihydro-6-methoxy-2-phenyl - 1- naphthyl)phenoxy]ethyl) pyrrolidine hydrochloride(Ui 1,1OOA)or of two related antiestrogens, Cis{3-Ip-(1 ,2,3,4-tetrahydro-6-methoxy-2-phenyl-1 -naphthyl)phenoxy}-1 ,2-propanediol) (U23,469) and a-(4-pyrrolidinoethoxy)phenyl-4-methoxy-a'-nitrostilbene (Cl-628) did not alter the activity fromthat of the diestrus control, and administration of antiestrogenalong with estradiol blocked estradiol stimulation nearly completely. Tumor peroxidase activity was stimulated 5-fold byestradiol (25 fLg)and 3-fold by antiestrogen (250 @tg1-{2-[p-(3 ‘4-dihydro-6-methoxy-2-phenyl-1 -naphthyl)phenoxyjethyl)pyrrolidmnehydrochloride) above the levels in tumors of maturecycling animals. With concomitant administration of antiestrogen and estradiol, stimulation of peroxidase was reduced tothe level seen with antiestrogen alone. In uteri of mammarytumor-bearing rats, some differences were seen. While estradiol significantly increased uterine weight and the activity ofuterine G6PD (expressed on a tissue weight or mg proteinbasis), the activities of malic enzyme and peroxidase were notincreased by estradiol above the levels seen in uteri of cyclinganimals. Ovariectomy and antiestrogen treatments, whichmarkedly reduced uterine weight, suppressed the activities of
these three enzymes far below the control level. When estradiolwas administered along with antiestrogen, uterine weights andmalic enzyme activity remained depressed. Estradiol stimulation of G6PD was reduced by antiestrogen, and uterine peroxidase activity returned to the high control level in antiestrogen plus estradiol-treated animals.
Hence, although antiestrogens and estrogens both reducegrowth of R3230AC mammary tumors, the results indicate thattheir effects on tumor enzyme activities differ. Also, althoughthe growth of both tumor and uterus is depressed by antiestrogen administration to mature cycling rats, the effects of antiestrogens on the activity of the same enzyme in tumor and uterusare frequently different.
INTRODUCTION
Synthetic estrogens and, more recently, antiestrogens havebeen used in the treatment of human breast cancer (4, 12, 13).The effectiveness of such endocrine therapy is positively correlated with the presence and content of estrogen receptors inmammary tumors; however, not all estrogen receptor-containing tumors are responsive to hormonal or antihormonal therapy(18, 23). Considerable current interest has focused on thedevelopment and exploitation of several mammary tumor systems in experimental animals that may serve as models forunderstanding the spectrum of hormone sensitivity of humanbreast cancers.
The R3230AC transplantable mammary tumor of the Fischer344 rat, one such experimental animal model, was characterized initially by Hilf et a!. (17). This mammary adenocarcinomawas found to grow equally well in ovariectomized or intacthosts, but continuous administration of high levels of estrogenor prolactin slowed the growth of these tumors (14, 16). Recently, our laboratory has shown that treatment with a varietyof nonsteroidal antiestrogens also markedly depresses thegrowth of these ovarian-autonomous tumors (28).
Since growth of this mammary tumor is ovarian autonomousyet estrogen responsive, our aim in this study has been 2-fold:first, to examine and compare the effects of antiestrogens andestradiol on the activity of several enzymes known to be modulated by estrogen (2, 16), namely, G6PD,3 NADP-dependentmalic dehydrogenase, called malic enzyme (L-malate:NADPoxidoreductase, decarboxylating), a-GPD, and peroxidase(22); and second, to compare the effects of antiestrogens and
, Supported by NIH Grants USPHS CA 181 19 from the National Cancer
Institute and HD 06726 and HD 07028 from the National Institute of Child Healthand HumanDevelopment.Reported, in part, atthe 61st Annual Endocrine SocietyMeeting, June 1979 (19).
2 To whom requests for reprints should be addressed at Department of
Physiology and Biophysics, 524 Burrill Hall, University of Illinois, Urbana. Ill.6 1801.
Received February 19. 1980; accepted May 22, 1980.
3 The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase
Antiestrogen Modulation of Mammary Tumor and Uterine Enzymes
estradiol on these enzymatic activities in mammary tumors anduteri of R3230AC tumor-bearing animals to enable a comparison of hormonal and antihormonal action in these 2 estrogensensitive tissues.
MATERIALS AND METHODS
R3230AC Rat Mammary Tumors. Forty-day-old femaleFischer 344 rats (from Charles River Breeding Laboratories,Inc., North Wilmington, Mass.) received s.c. axiliary transplantsof R3230AC tumor via a sterile trocar technique under lightsodium pentobarbital anesthesia. Twenty-one-day posttransplanted R3230AC tumors were removed from donor rats (kindlysupplied by Mason Research Institute, Worcester, Mass.),minced, and kept in iced sterilized Medium 199 (containingpenicillin and streptomycin) during tumor transplantation.Groups of rats either were ovariectomized 3 days before redelving tumor transplants or intact rats received vehicle (0.5 mlof 0.1 5 M NaCI containing 4% ethanol), various antiestrogens[U23,469 (100 jsg), Ui 1,100A (100 or 250 pg), and Cl-628(100 pig)], or estrogen plus antiestrogen (25 @tgestradiol plus100 or 250 ,zgU11,1OOA).The various compounds were givens.c. daily (in 0.5 ml 0.1 5 M NaCI containing 4% ethanol) for 25days beginning on the day of tumor transplantation. Tumorpalpation started 1 week after tumor transplantation and continued twice a week during the treatment period. Tumor sizewas measured in 2 dimensions (length x width) with calipers(28). At 25 days after tumor transplantation, the R3230ACtumor-bearing rats were sacrificed by decapitation, and theirmammary tumors and uteri were collected, quickly frozen ondry ice, and stored in a freezer at —85°until assayed. Controlrats were either sacrificed at diestrus or without regard to theirstage of the estrous cycle, as indicated in the chart legends.
Chemicals and Reagents. All enzyme substrates, NADP,NADH, and 17f@-estradiolwere obtained from Sigma ChemicalCo., St. Louis, Mo. The antiestrogens U23,469 (Isomer A) andUi 1,1OOA(nafoxidine HCI)were kindly provided by the UpjohnCo., Kalamazoo, Mich., and Cl-628 was kindly provided by theParke, Davis & Co., Ann Arbor, Mich.
Enzyme Activity Assays. R3230AC tumorsand uteri werehomogenized (100 mg/mI) in 0.01 M Tris-HCI buffer, pH 7.4,and assays for 3 of the enzymes were performed on thehomogenate supernatant (20,000 x g for 30 mm). Assayswere carried out under conditions showing zero-order kinetics,and the absorbance change at 340 nm due to the productionof NADPH or the oxidation of NADH was monitored. Theenzymes monitored were G6PD, by the method of Glock andMcLean (11), a-GPD, by the procedure of Beishenherz et a!.(3) modified by using dihydroxyacetone phosphate as thesubstrate and measuring the oxidation of NADH as describedby Cohen and Hiif (6), and NADP-malate dehydrogenase, decarboxylating (malic enzyme) by the method Ochoa et a!. (25).
Tumor peroxidase activity was solubilized, as described byLyttle and DeSombre (22), by rehomogenizing the sediment(40,000 x g for 30 mm) with 10 mMTris-HCI, pH 7.2, containing 1.0 M CaCI2. Uterine peroxidase activity was solubilizedwith 10 mM Tris-HCI, pH 7.2, containing 0.5 M CaCl2. Solubilization of peroxidase activity of uterus and tumor was monitored with different concentrations of CaCI2and was found tobe complete with the CaCl2 concentrations stated above. Noperoxidase activity was found in the 40,000 x g supernatant
fraction. Peroxidase activity was determined exactly as described by Lyttle and DeSombre (22). The assay mixture (3.0ml total volume) contained 13 mM guaiacol, 0.3 mM H202, andan aliquot of sample extract. The rate of oxidation of guaiacolwas measured in the presence of H202 as indicated by achange in absorbance at 470 nm.
Statistics. The significance of differences between treatmentgroups and the control group was examined by Student's ttest.
RESULTS
Eftects of Estrogenand Antiestrogen on Enzyme Activitiesin R3230AC Mammary Tumors. Since in previous publications(19, 28) we found that antiestrogens and estrogen diminishedthe rate of R3230AC mammary tumor growth, we decided todetermine if these compounds affected R3230AC tumor enzyme activities in a parallel manner. For these studies, eitherintact Fischer 344 rats were treated with antiestrogen (250 @tg),estradiol (25 ftg), or vehicle control daily for 24 days beginningon the day of tumor transplantation or tumors were transplantedinto females that had been ovariectomized 3 days prior to thetime of tumor transplantation. When harvested at 25 days aftertransplantation, tumors were much smaller in antiestrogentreated and estradiol-treated host animals, while tumor sizewas similar in control and ovariectomized hosts. These tumorgrowth profiles have been published previously (28). The effects of these hormonal-antihormonal treatments on the activities of several metabolic enzymes in mammary tumors areshown in Chart 1. Estradiol increased the activity of G6PD andmalic enzyme 2-fold (expressed on a tissue weight or mgprotein basis) and decreased the activity of s-GPD 2-fold, inkeeping with previous reports (14—17). Antiestrogen treatment(Cl-628, U23,469, or Ui 1,1OOA) and ovariectomy did notchange the activity of these enzymes from that of the diestruscontrol. The concomitant administration of antiestrogen alongwith estradiol greatly diminished the estradiol-stimulated increase in G6PD activity and completely blocked the increasein malic enzyme activity seen with estradiol alone (Chart 1). Hilf
Chart 1. Effects of antiestrogen and/or estradiol treatment or of ovariectomyon enzyme activities of R3230AC mammary tumors. Fischer 344 host rats (8 to12/group) received antiestrogen [Cl-628 (Cl). U23,469 (U-23), or Ui 1,1OOA(UA)1(250 @gs.c. in 0.15 M NaCI daily), estradiol (E2)(25 ;ugs.c. in 0.9% NaCIsolution daily), estradiol (25 tug)plus antiestrogen (250 @.tgU11,1OOA),or vehicle(0. 15 M NaCI) as control (C) for 24 days be9inning on the day of tumortransplantation, or tumors were transplanted into host rats that had been ovariectomized (OVX) 3 days prior to the time of tumor transplantation. All tumorswere harvested at 25 days after transplantation [controls were in diestrus at 25± 1 (SE.) days after tumor transplantation), and the 20,000 x g supernatantfraction of tumors was assayed for G6PD, malic enzyme, and a-GPD activities.Numbers in columns, number of different tumors assayed; columns, mean; bars,SE.; ‘, values significantly different from the controls at p < 0.05.
(15) has reported a similar depression of estradiol-stimulatedincreases in these enzymes by the antiestrogen MER-25.
Peroxidase is an enzyme whose activity has been shown tobe markedly increased by estrogen in hormone-dependentDMBA-induced rat mammary tumors and in uteri of severalanimal species (8, 21). Hence, we examined the effects ofhormonal manipulation on peroxidase activity in this ovarianautonomous mammary tumor. Our findings are reported inChart 2.
Peroxidase activity was found to be low in mammary tumorsof control rats and was increased 5-fold following treatmentwith a high dose (25 @.sg)of estradiol. Antiestrogen treatmentincreased peroxidase activity 3-fold, and when antiestrogenwas administered along with estradiol, mammary tumor peroxidase activity was only stimulated 3-fold, namely, to the levelseen with antiestrogen alone. Ovariectomy had no effect on thelevel of peroxidase activity in tumors (Chart 2).
Effects of Estrogen and Antlestrogen on Enzyme Activitiesin Uteri. In uteri of mammary tumor-bearing animals, dailyadministration of estradiol (25 pg/day) increased uterineweight above that of cycling females, while ovariectomy orantiestrogen administration resulted in a marked decrease inuterine size (Chart 3). When antiestrogen was administeredwith estrogen, uterine weights were greatly depressed, but notto the extent seen with antiestrogen alone (Chart 3). Uterineweights were consistently lower in antiestrogen-treated animalsthan in ovariectomized Fischer rats, although body weights didnot differ significantly between treatment groups. This differsfrom the situation in Sprague-Dawley rats bearing DMBA-induced mammary tumors in which similar daily antiestrogenadministration decreased uterine weights below those of controls, but never to the extent seen in ovariectomized animals(27). This may suggest a significant adrenal gland contribution
in these Fischer 344 rats, a possibility that we have yet toexplore.
Chart 4 reports the effects of these endocrine manipulationson the activities of various enzymes in uteri. Estradiol increasedthe activity of uterine G6PD, while ovariectomy or antiestrogenadministration significantly depressed this activity. These depressive effects of ovariectomy and antiestrogen on G6PDactivity in uteri are in contrast to the lack of effect of thesetreatments on G6PD activity in R3230AC mammary tumors. Asseen in Chart 4, when antiestrogen treatment (100 or 250 @tgUi 1,1OOAper day) was combined with estrogen treatment (25@sg/day),uterine G6PD activity was found to be equivalent tothe level seen in control uteri.
The activity of another uterine enzyme, malic enzyme, appeared to be increased following estrogen treatment, but notto a level that was statistically different(p < 0.05) from controls(Chart 4). Nevertheless, ovariectomy and antiestrogen treatment significantly depressed malic enzyme activity in uteri.Malic enzyme activity also remained depressed when antiestrogen treatment was combined with estrogen treatment.
Peroxidase activity (Chart 5) was found to be high in uteri ofcontrol cycling animals, and it was not stimulated further bythe administration of estradiol. Ovariectomy or antiestrogentreatment almost completely abolished peroxidase activity, but,when antiestrogen was administered together with estradiol,uterine peroxidase activity returned to the high level seen incontrol and estradiol-treated females (Chart 5).
DISCUSSION
These results indicate significant differences in the responses of enzymes of the R3230AC mammary tumor to estrogen and antiestrogen, even though both endocrine treatmentsgive similar depression of the growth rate of these mammarytumors. Also, although the growth of uterus as well as tumor isdepressed by antiestrogen, the effects of antiestrogen on theactivity of the same enzyme in tumor and uterus are frequently
Chart 3. Effects of antiestrogen and/or estradiol treatment or of ovariectomyon uterine wet weight. Fischer 344 host rats received antiestrogen Ui 1,1OOA(UA) (250 ,zgs.c. in 0. 15 M NaCI daily), estradiol (E2)(25 @gs.c. in 0. 15 M NaCIdaily), antiestrogen (100 or 250 p9) plus estradiol (25 ig), or vehicle (0. 15 MNaCI) as control for 24 days beginning on the day of tumor transplantation, oranimals were ovariectomized (OVX) 3 days prior to tumor transplantation. Uteriwere excised at 25 days after tumor transplantation and weighed. Numbers incolumns, number of individual uteri assayed; columns, mean; bars, SE. ; ‘.values significantly different from the controls (uteri of mature cycling animals) atp<0.05.
MAMMARY TUMORPEROXIDASEACTIVITY
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- CONTROL25@qE@ OVX 2@0@UAi0c@gUA2@4@gUA
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Chart 2. Effects of antiestrogen and/or estradiol treatment or of ovariectomyon peroxidase activity of R3230AC mammary tumors. Fischer 344 host ratsreceived antiestrogen U 11,1OOA(UA)(250 @igs.c. in 0. 15 M NaCI daily), estradiol(E2)(25 jig s.c. in 0. 15 M NaCIdaily), antiestro9en (100 or 250 sag)plus estradiol(25 rig). or vehicle (0. 15 M NaCI) as control for 24 days beginning on the day oftumor transplantation. or tumors were transplanted into host rats that had beenovariectomized (OVX) 3 days prior to tumor transplantation. All tumors wereharvested at 25 days after transplantation and were assayed for peroxidaseactivity as described in ‘‘Materialsand Methods.‘â€Numbers in columns, numberof individual tumors assayed; columns, mean; bars, SE.; ‘. values significantlydifferent from the controls at p < 0.05.
Antiestrogen Modu!ation of Mammary Tumor and Uterine Enzymes
There is considerable evidence that the enzyme peroxidaseis a meaningful marker of estrogen-induced responses in reproductive tissues. Peroxidase has been identified histochemically in the normal rat mammary gland (1 , 26) and in theDMBA-induced mammary tumor (7), and it has been shown tobe localized in epithelial cells. We believed, therefore, thatperoxidase might serve as an appropriate marker of estrogenresponsiveness in the R3230AC tumor, which is epithelial innature. Our results demonstrate a low level of peroxidaseactivity in control tumors that is stimulated 5-fold by estrogentreatment. Ui 1,100A was a weak agonist in this assay, sinceit stimulated peroxidase activity over 2-fold. Interestingly, whenUi 1,100A was administered together with estradiol, it antagonized the ability of estradiol to stimulate peroxidase activity inmammary tumors, in contrast to its lack of antagonism ofestradiol action at similar doses in the uterus. In some tissuessuch as rat uterus, some effects of antiestrogen versus estradiol have been attributed to differential stimulation of differentcell types by the 2 agents (5). In this R3230AC tumor, whichconsists almost exclusively of epithelial cells, the effects ofestradiol and antiestrogen could not be explained by differentialcell stimulation.
Peroxidase activity was found to be high in uteri of controlcycling rats, and, while not being significantly increased by theadministration of a high (25 gig)estradiol dose, uterine peroxidase activity was drastically reduced by ovariectomy or antiestrogen. These findings are in agreement with those of DeSombre and Lyttle (9), who have shown that, while total uterineperoxidase activity increases during estrus, peroxidase activitydoes not change in uteri during the cycle when expressed ona tissue weight basis. In the present study, the low level ofperoxidase activity in uteri of ovariectomized or antiestrogentreated females suggests that this enzyme is very sensitive toendogenous hormone, such that the endogenous physiologicallevels of estrogen in cycling rats maximally stimulate its activity.However, while antiestrogen (250 zg, Chart 5; or 100 jzg, notshown) was able to effectively antagonize the endogenousestrogen in control cycling rats, as monitored by inhibition ofperoxidase, similar antiestrogen doses were not able to antagonize pharmacological (25 @sg/day)doses of estradiol. Presumably, the ratio of antiestrogen to estrogen would have to bemuch higher than the one we used to prevent estrogen fromstimulating uterine peroxidase activity. However, this ratio ofantiestrogen to estrogen was effective in reducing uterineweight and uterine malic enzyme activity (Charts 3 and 4).
Of note are the marked quantitative differences in the basalactivities and stimulated levels of peroxidase in mammary tumorand uterus. Although the activities of G6PD and malic enzymewere similar (on a protein, DNA, or tissue weight basis) inuterus and R3230AC tumor, the activity of peroxidase inR3230AC tumors was only 0.5% that seen in uteri from thesame control animals. A comparison between the levels ofenzyme activities in the R3230AC tumor (this paper) and thosereported for the DMBA-induced mammary tumor (8) revealsthat there is 10 times as much peroxidase activity in the DMBAtumor as in the R3230AC tumor. High levels of peroxidaseactivity are also found in human breast cancer samples, and aqualitative correlation between the presence of estrogen receptor and peroxidase activity has been shown in human breasttumors (10, 21).
The results we have found here are consistent with the
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Chart 4. Effects of antiestrogen and/or estradiol treatment or of ovariectomyon enzyme activities of uteri of R3230AC tumor-bearing animals. Fischer 344host rats (8 to 10/group) received antiestrogen U11,1OOA(UA) (250 @igs.c. in0.15 M NaCI daily), estradiol (E2)(25 @ags.c. in 0.15 PANaCI daily), antiestrogen(100 or 250 @&g)plus estradiol (25 sag),or vehicle (0. 15 MNaCi) as control (CONT)for 24 days beginning on the day of tumor transplantation, or animals wereovariectomized (OVX) 3 days prior to tumor transplantation. Uteri were excisedat 25 days after tumor transplantation and the 20,000 x g supernatant fractionwas assayed for G6PD and malic enzyme activities. Numbers in columns, numberof individual uteri assayed; columns, mean; bars, SE.; ‘, values significantlydifferent from controls at p < 0.05.
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Chart 5. Effects of antiestrogen and/or estradiol treatment or of ovariectomyon peroxidase activity of uteri of R3230AC tumor-bearing animals. Symbols andtreatment of animals exactly as described in the legend to Chart 2. Uteri wereexcised and assayed at 25 days after tumor transplantation. Numbers in columns,number of Individual uteri assayed. Columns, mean; bars, SE.; ‘. values significantly different (p < 0.05) from the control uteri.
different. Thus, we found that Ui 1,1OOAsuppressed uterineweight and the activities of uterine peroxidase, G6PD, andmalic enzyme, while identical doses of this antiestrogen actually increased the activity of peroxidase in mammary tumorand was without effect on the activity of mammary tumor G6PDand malic enzyme. Differing effects of the antiestrogen CI-628on enzymatic activities in brain, pituitary, and uterus have alsobeen reported (20).
In mammary tumor, the effects of estradiol and antiestrogenon enzyme activities frequently differed also. Estradiol increased the activity of 3 of the enzymes studied, namely,G6PD, malic enzyme, and peroxidase in mammarytumor; incontrast, ovariectomy and antiestrogen treatment were withouteffect on these enzyme activities in mammary tumors. In addition, while a-GPD activity was suppressed in mammary tumorsby estradiol, as reported previously by Hilf et a!. (16), thisenzyme activity was also unaffected by ovariectomy or administration of antiestrogen.
suggestion that the responsiveness of a target organ to estrogen can be correlated with the presence of the estrogenreceptor. The R3230AC tumor contains estrogen receptor witha high affinity for estradiol [Kd = 2 x 10 10M (24, 29), similarto that found in DMBA tumors (24) and normal lactating mammary gland (29)], but receptor levels are only 10 to 30% thelevel found in DMBA tumors (24, 27, 28) or lactating mammarygland (29). In addition, estrogens as well as antiestrogensinteract with the cytoplasmic receptor in R3230AC tumors andmove the receptor into the nuclear compartment (28). It hasbeen proposed that the estrogen sensitivity, but lack of ovariandependence, of the R3230AC tumor may be associated withthis low estrogen receptor level (24). Nonetheless, the observation that estrogen and antiestrogens interact with estrogenreceptors in R3230AC tumors and modulate enzyme activitiesand tumor growth rate indicate that this tumor is clearly estrogen responsive, although not ovarian dependent, for its growth.This tumor has also been shown to respond to estrogen withlactation-like morphological and biochemical changes and tobe prolactin sensitive. However, the possibility that the differingenzyme results in tumor and uterus might be due to prolactinseems unlikely. At the doses used in this study, the variousantiestrogens did not alter serum prolactin levels (28). In addition, Hilf et a!. (17) have reported that estrogen stimulation oftumor G6PD and malic enzyme occurred in the presence orabsence of high levels of prolactin.
The present results indicate clear differences in the sensitivities of several enzymes to estrogen and antiestrogen andemphasize that the effects of antiestrogens on the activity ofthe same enzyme in tumor and uterus are frequently different,although the growth of both tumor and uterus is similarlydepressed by antiestrogen treatment. These considerationswould seem important in interpreting the varied effects ofantiestrogens, when given therapeutically, in different estrogentarget tissues.
ACKNOWLEDGMENTS
We are grateful to Dr. A. Bogden of the Mason Research Institute for supplyingus with donor rats bearing R3230AC tumors and to Parke, Davis & Co. andUpjohn Co. for providing us with antiestrogens. We also thank Dr. Ten-unTsai forher contributions to this study.
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