Effect of Estradici and Prolactin on Galactosyltransferase...

[CANCER RESEARCH 38, 2077-2083, July 1978]0008-5472/78/0038-0000$02.00

Effect of Estradici and Prolactin on Galactosyltransferase anda-Lactalbumin Activities in Rat Mammary Gland and MammaryTumor1

Clement Ip and Thomas L. Dao2

Department of Breast Surgery and Breast Cancer Research Unit, Roswell Park Memorial Institute, Buttalo, New York 14263

ABSTRACT

We have reported the conditions for the in vivo stimulation of galactosyltransferase and a-lactalbumin activitiesin the mammary gland of virgin ovariectomized rats byexogenous hormones. Administration of 1 /tg estradiciand 1 mg prolactin daily for 6 days produces a maximaleffect, provided the mammary tissue has already beenprimed by a combination of 1 fig estradici and 3 mgprogesterone daily for 14 days to promote cellular proliferation. Although the data suggest that prolactin may bethe key hormone that triggers the expression of bothproteins, estrogen is necessary, especially for maximalstimulation of a-lactalbumin activity. A high dosage ofestradici (10 /Â¿g/day),however, results in an inhibitoryresponse.

Of the 15 dimethylbenz(a)anthracene-induced mammary tumors studied, only 4 have detectable a-lactalbumin activity, although they all contain different levels ofgalactosyltransferase. Estradici and prolactin administration causes a slight increase in the level of both proteinsin these 4 tumors, but the remaining 11 tumors areunresponsive to the effect of this hormonal combination.There does not seem to be an absolute correlation between the estrogen-binding capacity and the productionof a-lactalbumin. However, preliminary data show that ofthe four tumors with the highest estrogen-binding capacity, three are positive for a-lactalbumin. The inability ofmost of these tumors to synthesize a-lactalbumin, evenwhen the hosts have undergone pregnancy and lactation,suggests that in the majority of cases the gene coding forthis tissue-specific protein is probably not expressed as aresult of malignant transformation.

INTRODUCTION

Lactose synthesis in the mammary gland is catalyzed by2 structurally and functionally dissimilar proteins, galactosyltransferase and a-lactalbumin (4, 5). The normal functionof galactosyltransferase is to catalyze the transfer of galactose to A/-acetylglucosamine in the synthesis of oligosac-charide prosthetic groups of glycoproteins. a-Lactalbumin,a protein that is unique to the mammary tissue, modifiesthe acceptor specificity of galactosyltransferase from N-acetylglucosamine to glucose so that lactose synthesis canbe achieved under physiological conditions (3).

1 This investigation was supported by Grant CA 14812-03, awarded by the

National Cancer Institute, Department of Health, Education and Welfare.2 To whom requests for reprints should be addressed, at Roswell Park

Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14263.Received Octobers, 1977; accepted April 4, 1978.

The appearance of galactosyltransferase and a-lactalbumin during different stages of pregnancy and lactation inrodents has been examined by several groups of workers(18, 23, 28, 40). The galactosyltransferase activity starts toemerge during the latter half of gestation and increasesonly moderately during lactation. In contrast, a-lactalbuminis not synthesized in sufficient quantity to support lactoseformation until just prior to parturition, at which time thelevel rises sharply in the mammary gland. It is not fullyunderstood at this time what physiological factors aredirectly responsible for the genotypic expression of these 2proteins in vivo, although prolactin is widely accepted asthe lactogenic hormone. The sudden surge in a-lactalbuminproduction has been attributed to the release of inhibitionof its synthesis by the decrease in circulating progesteronenear parturition (19, 26).

Recently, attention has been focused on the ability ofmammary carcinoma to synthesize milk proteins such ascasein and a-lactalbumin and the influence of hormones onthis parameter. The synthesis of gene products as a resultof hormonal stimulation should reflect the hormonal responsiveness of the tumor.

In this study we report the in vivo stimulation of bothgalactosyltransferase and a-lactalbumin activities in themammary gland of mature virgin female rats by exogenousestradici and prolactin and the influence of hormones onthese proteins in the DMBA3-induced mammary tumors.The term "induction" as used in the text refers strictly to an

increase in protein activity and does not specify the mechanism involved.

MATERIALS AND METHODS

Hormone and Drug Preparation. Estradici and progesterone (Sigma Chemical Co., St. Louis, Mo.) were first dissolved in ether and then mixed with commercial olive oil.The ether was subsequently evaporated with nitrogen.Ovine prolactin (NIAMDD, Hormone Distribution Program)was dissolved in phosphate-buffered saline (pH 8.0) (50 mwsodium phosphate in 0.9% NaCI solution). Trilaform(aqueous solution of perphenazine) was purchased fromSchering Corp., Kenilworth, N. J. 2-Bromo-a-ergocryptine(CB-154; Sandoz Pharmaceuticals, East Hanover, N. J.) wasprepared by dissolving the ergot first in ethanol and thendiluting it with 0.9% NaCI solution. Hormones and prolactinrelease inhibitor (CB-154) were administered s.c. in 0.2-mlaliquots. The dosages and the schedule of injections aregiven in detail in each table.

3 The abbreviations used are: DMBA. 7,12-dimethylbenz(a)anthracene;

NIAMDD, National Institute of Arthritis, Metabolism, and Digestive Diseases.

JULY 1978 2077

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C. Ip and T. L. Dao

Animals. Ovariectomized Sprague-Dawley rats (240 g)

were used in the experiments concerning the in vivo hormonal induction of galactosyltransferase and a-lactalbu-min. Mammary tumors were induced in female Sprague-

Dawley rats by i.g. instillation of 20 mg of DMBA in sesameoil when the animals were 50 to 55 days old. Rats werepurchased from A. R. Schmidt, Madison, Wis.

Assay of Galactosyltransferase and a-Lactalbumin Ac

tivities. Mammary tissue and tumor samples were droppedinto liquid nitrogen immediately after excision from theanimals and stored at -70Â° until ready for assay. Galacto

syltransferase and (v-lactalbumin activities were assayed by

the method described by Vonderhaar ef al. (46) with modifications. Pulverized mammary or tumor tissue was homogenized in an ice-cold buffer containing 0.02 M Tris-HCI (pH7.4), 0.01 M MgCL, and 0.001 M /3-mercaptoethanol with aPotter-Elvehjem homogenizer. The crude homogenate wascentrifuged at 1000 x g for 5 min to remove cell debris.Approximately 5% of the galactosyltransferase activity wasfound in the cell debris fraction. Fifty Â¿tlof the supernatantfraction were used for the assay.

The reaction mixture contained 2 /xmol of Tris-HCI buffer(pH 7.4), 1 Â¿imolof MnCI2, 40 nmol of UDP-[U-14C]galactose

[approximately 60,000 dpm (New England Nuclear, Boston,Mass.)], 0.1% Triton X-100, and 0.2 /Â¿molof UTP to inhibitthe UDP-galactose hydrolase activity (7). The total volume

of the reaction mixture was 100 n\. This concentration ofUTP had no perceptible effect on either galactosyltransferase or Â«-lactalbumin activities but resulted in a substantial

reduction in hydrolase activity.For determination of galactosyltransferase activity, 2

Â¿Â¿molof /v-acetylglucosamine were used as the acceptor togenerate /V-[14C]acetyllactosamine. Â«-Lactalbumin activity

was estimated in the presence of an excess of bovine milkgalactosyltransferase (5 milliunits; Sigma) and 2 /nmol of D-glucose as acceptor to form [14C]lactose. A standard curvewas generated with increasing amounts of bovine a-lactal-

bumin to ensure that the reaction was in the linear portionof the curve with respect to a-lactalbumin concentration

(3). Assays for both reactions were run in triplicate. Tocorrect for the production of [14C]galactose resulting fromendogenous hydrolysis of UDP-[14C]galactose, a control

reaction was included for each sample in the absence ofeither acceptor. Approximately 0.7 to 3% of the UDP-galac

tose was hydrolyzed during the incubation period.Incubation of the reaction mixture was carried out at 37Â°

in a Dubnoff shaker for 30 min. It was stopped by cooling inice and subsequently adding 100 /Â¿Iof cold water. Thecontent of each tube was quickly passed through a column(0.5 x 4 cm) of Bio-Rad AG 1-X2 aniÃ³n exchange resin in the

chloride cycle. Reaction tubes were washed with 0.5 ml ofwater that was then transferred to the columns. Neutralsugars on the column were eluted with another 1 ml ofwater directly into scintillation vials. Radioactivity in theeluate was measured with 10 ml of ACS (Amersham/SearleCorp., Arlington Heights, III.) by liquid scintillation counting. Recovery of neutral sugars from the column was over98% with no elution of UDP-galactose.

Â«-Lactalbumin has been shown to inhibit the activity ofgalactosyltransferase with /V-acetylglucosamine as substrate (3). The 1000 x g homogenate used for the assay of

galactosyltransferase also contains a-lactalbumin. Thus,

the values obtained for galactosyltransferase by thismethod may be underestimated.

Estrogen Receptor Assay. Quantitative determination ofestrogen receptor was carried out by the method previouslydescribed (33). The pulverized tumor (0.5 g) was homogenized in 1 ml of buffer (0.01 M Tris, 0.001 M EDTA, 0.01 Mthioglycerol, and 10% glycerol, pH 7.8). The cytosol wasisolated and incubated with 5 nM [2,4,6,7-3H]estradiol (NewEngland Nuclear) for 4 hr at 0Â°in the presence and absenceof nafoxidine. A dextran-coated charcoal pellet was used to

remove excess steroid. The samples were layered on top of10 to 30% sucrose gradients and centrifuged for 16 hr at50,000 rpm with an SW-60 rotor in a Beckman LS-50

centrifuge. The fmol of estradiol bound per mg of proteinwere determined in the 4S and 8S regions of the gradient.

Determination of Serum Prolactin. Serum prolactin wasdetermined with the kit obtained from NIAMDD. The reference prolactin standard supplied was RP-1 with a biological

potency of 11 lU/mg. Lactoperoxidase (Calbiochem, SanDiego, Calif.) was used for iodination (12). Serum prolactinwas assayed by the double-antibody radioimmunoassay

procedure recommended by the directions supplied withthe NIAMDD kit with 2 modifications. The concentration of[125l]prolactin was increased to 60,000 cpm, and the incu

bation time with the first antibody was reduced to 24 hr.Serum samples were assayed at 2 dilutions each in triplicate.

Protein and DMA Determinations. Protein was determined as described by Lowry ef al. (20) with crystallizedbovine serum albumin (Schwarz/Mann, Orangeburg, N. Y.)as standard. DNA was estimated according to the methodof Burton (6).

RESULTS

Galactosyltransferase and a-Lactalbumin Activities inNormal Lactating Mammary Gland. Table 1 shows thegalactosyltransferase and a-lactalbumin activities in the

mammary gland of virgin, pregnant, and lactating rats.Levels of galactosyltransferase and a-lactalbumin weremuch higher at parturition than that found in midpreg-nancy. This is in agreement with the observations reportedby other investigators (18, 23). Both activities continued torise after initiation of lactation.

Effect of in Vivo Hormone Administration on the Stimulation of Galactosyltransferase and a-Lactalbumin Activi-

Table 1Galactosyltransterase and a-lactalbumin activities in mammary

gland of virgin, pregnant, and lactating ratsNo. of Galactosyltrans-

Physiological state rats ferase" a-Lactalbumin"

MaturevirginMidpregnantParturientLactating,

fourthday64651.5

Â±0.1e2.7Â±0.224.8Â±2.133.1Â±2.5NilNil15.5

Â±1.223.6Â±2.0

" nmol of A/-acetyllactosamine formed per mg protein per 30

min.b nmol of lactose formed per mg protein per 30 min.c Mean Â±S.E.

2078 CANCER RESEARCH VOL. 38



Hormonal Control of o-Lactalbumin in Vivo

ties in Ovariectomized Rats. All rats were ovariectomizedin this experiment, and they were injected with 1 /xgestradici and 3 mg progesterone daily for 14 days topromote cellular proliferation in the mammary gland. Table2 summarizes the results. Priming of the mammary tissueby steroid hormones was essential for the detection offunctional activity induced by repeated injections of estradici and prolactin. Submaximal stimulation of galactosyl-transferase and a-lactalbumin activities was obtained whenintact female rats were used, possibly due to the luteo-trophic effect of exogenous prolactin (results not shown).

When ovariectomized rats were primed by a combinationof estradiol and progesterone for 14 days, subsequentadministration of 1 Â¿c.gestradiol and 1 mg ovine prolactindaily for 6 days (Group 2) resulted in a marked increase inboth galactosyltransferase and a-lactalbumin levels. Thiswas in contrast to the much lower enzyme activities obtained in Group 1, in which the animals received no estradiol and prolactin treatment. The total DNA content in the 6abdominal-inguinal glands from rats in Group 2 was 6.4 Â±0.5 (S.E.) mg/100 g body weight, as compared to 8.1 Â±0.5mg obtained at parturition and 1.8 Â±0.2 mg from untreatedvirgin control rats. When progesterone injection was continued throughout the 20-day period (Group 3), the activitiesof both proteins were greatly reduced.

The role of individual hormones was also studied in thisseries of experiments. Administration of 1 ^9 estradiol and3 mg progesterone for 14 days followed by 6 days of 1 /itgestradiol alone (Group 4) resulted in only a moderateenhancement of galactosyltransferase and a-lactalbuminactivities. To minimize the effect of estradiol-stimulatedrelease of endogenous prolactin from the pituitary, rats

were also given injections of 2-bromo-a-ergocryptine fromDays 15 to 20 (Table 2, Footnote a). The concentration ofcirculating prolactin in this group was 34 Â±4 ng/ml,compared to the basal level of 29 Â±3 ng/ml in normalcycling female rats.

Injection of 1 mg of prolactin alone from Days 15 to 20(Group 5) led to a submaximal response in a-lactalbuminactivity when compared to the combination of estradiol andprolactin (Group 2, p < 0.05). This finding suggests that,although prolactin is the key hormone for the induction ofa-lactalbumin, the presence of estradiol is essential foroptimal stimulation. On the other hand, prolactin aloneappeared to be as effective as estradiol and prolactin in thestimulation of galactosyltransferase (p > 0.05, Group 2versus Group 5). Administration of progesterone togetherwith either 1 /Â¿gof estradiol or 1 mg prolactin from Days 15to 20 also resulted in an inhibitory response (Groups 6 and7).

The concentration of estradiol seems to be critical in theinduction of both proteins. This is evidenced by the resultsfrom Group 8, which show that administration of a highdose of estradiol (10 Â¿ig)together with prolactin actuallyproduced an inhibitory response. As expected, exogenousprolactin can be replaced by perphenazine injection (Group9), which is known to increase prolactin level in the circulation. Serum prolactin concentration in this group wasfound to be 306 Â±42 ng/ml.

Effect of Estradiol and Prolactin Administration on Galactosyltransferase and Â«-LactalbuminActivities in DMBA-induced Mammary Tumors. Experiments were carried outto determine: (a) whether the DMBA-induced mammarytumors retain a-lactalbumin activity; (b) the responsiveness

Table 2Effect of hormones on the stimulation of galactosyltransferase and o-lactalbumin

activities in the mammary gland of ovariectomized ratsTreatment" (Days 15 to 20) Galactosyltrans-

Group No. of rats (dose/day) ferase* -Lactalbuminc

1010

10

10106

6

10

10

None1 /Â¿gestradiol + 1 mg

prolactin1 M9 estradiol + 1 mg

prolactin + 3 mgprogesterone

1 M9estradiol1 mg prolactin1 /Â¿gestradiol + 3 mg

progesterone1 mg prolactin + 3 mg

progesterone10 /Â¿gestradiol + 1 mg

prolactin1 /ugestradiol + 0.5 mg

perphenazine

5.6 Â±0.8"16.3 Â±1.5

3.4 Â±0.4e

8.2 Â±1.4e12.7 Â±1.5'3.1 Â±0.5"

3.8 Â±0.5*

6.5 Â±0.8e

14.9 Â±1.6'

0.8 Â±0.19.6 Â±0.9

1.8 Â±0.3e

2.2 Â±0.2e6.8 Â±0.8e1.2 Â±0.2"

1.1 Â±0.2*

3.7 Â±0.5e

8.8 Â±1.0'

" All rats were given injections of 1 /Â¿gestradiol plus 3 mg progesterone daily fromDays 1 to 14. With the exception of those in Group 9, rats in these groups were also giveninjections of 0.5 mg of 2-bromo-a-ergocryptine from Days 15 to 20 to suppress endogenous prolactin release. Animals were sacrificed on Day 21.

6 nmol of N-acetyllactosamine formed per mg protein per 30 min.c nmol of lactose formed per mg protein per 30 min.d Mean Â±S.E.e p < 0.05 compared to the corresponding value in Group 2.' p < 0.05, not significant compared to the corresponding value in Group 2.ap < 0.05 compared to the corresponding value in Group 4.* p < 0.05 compared to the corresponding value in Group 5.

JULY 1978 2079



C. Ip and T. L. Dao

of this parameter to hormonal stimulation; and (c) if there isany correlation between estradiol-binding capacity and thelevel of this activity. To study the effect of hormones oneach individual tumor, a biopsy sample of the tumor wastaken for galactosyltransferase and a-lactalbumin assaysand estradici receptor determination at the time of ovariec-tomy. The animals were then given injections of 1 /*gestradici and 1 mg prolactin daily for 6 days. Galactosyltransferase and a-lactalbumin assays were then performedon the remaining portion of the tumor. The results aresummarized in Table 3.

A total of 15 adenocarcinomas were obtained from 8 rats.Due to insufficient material in certain samples, the estrogen-binding assay was performed in only 10 tumors. Theestrogen receptor levels covered a wide range, varying from13 to 166 fmol of estradici bound per mg of cytosol protein.All 10 tumors showed a 4 to 5S component in the sucrosegradient analysis. Boylan and Wittliff (2) questioned thevalidity of this species and attributed the radioactivitybound to this region of the gradient to nonspecific bindingof the labeled estrogen to plasma proteins. We had thoroughly rinsed the tumor samples to eliminate as much ofthe blood contamination as possible. Moreover, the use ofnafoxidine as an antagonist should greatly minimize nonspecific binding to contaminating plasma components.

Of the 15 tumors studied for galactosyltransferase and a-lactalbumin activities, only 4 (in Rats 3, 6, 7, and 8) haddetectable a-lactalbumin activity, whereas various levels ofgalactosyltransferase were found in all tumors. Estradici

and prolactin administration led to a slight increase ingalactosyltransferase and a-lactalbumin activities in these4 tumors but had no appreciable effect in the rest. Tumorsfrom the same rat may differ in the production of a-lactalbumin. Although there did not seem to be an absolutecorrelation between the estrogen-binding capacity and theproduction of a-lactalbumin, in the 4 tumors with thehighest estrogen-binding capacity 3 contained a-lactalbumin.

Effect of Pregnancy and Lactation on Galactosyltransferase and a-Lactalbumin Activities in DMBA-inducedMammary Tumors. Nine rats bearing tumors were used inthis experiment, but only 4 completed pregnancy. They allhad small litters and showed signs of rejecting the pups. All4 dams were sacrified 3 days postpartum. Results of thegalactosyltransferase and a-lactalbumin activities in themammary tumors and the host mammary glands are presented in Table 4. A total of 6 tumors were examined, butonly 1 showed the presence of a-lactalbumin, although theyall contained different levels of galactosyltransferase. Interestingly, both protein activities in the host mammary glandsof all 4 dams were significantly lower than that found innormal control rats (refer to Table 1).

DISCUSSION

We have reported in this study the conditions for the invivo induction of galactosyltransferase and a-lactalbuminin the mammary glands of mature virgin female rats by

Table 3Effect of exogenous estradici and prolactin on galactosyltransferase and a-lactalbumin activities

in DMBA-induced mammary tumors of ovariectomized ratsA biopsy sample of the tumor was taken at the time of ovariectomy for estradici binding andgalactosyltransferase and a-lactalbumin assays. Animals were subsequently given injections of 1fj.g of estradici and 1 mg of prolactin daily for 6 days.

Rat

Pretreatment Posttreatment

No. of tumors

Estradiol binding"

Galactosyltransferase''

a-Lactalbu-minr

Galactosyltransferase

a-Lactalbumin

NDd

ND

ND13.3

1.061.52

1.431.47

NilNil

NilNil

1.171.86

1.311.17

NilNil

NilNil

20.3

ND30.9

ND24.766.2

2.45

0.750.83

0.950.730.98

0.75

NilNil

NilNilNil

3.88

0.840.75

0.980.840.88

1.36

NilNil

NilNilNil

58.176.2

0.873.14

Nil1.02

0.964.72

Nil1.45

781275.082.2166.03.710.801.171.33Nil0.875.160.922.622.08Nil1.33

" fmol of estradici bound per mg cytosol protein.b nmol of A/-acetyllactosamine formed per mg protein per 30 min.' nmol of lactose formed per mg protein per 30 min.d ND, not determined.





Table 4Galactosyltransferase and o-lactalbumin activities in the DMBA-

induced mammary tumor and the host mammary gland ofpostpartum rats

Host mammaryglandRat1234Litter

size4546Galactosyltransferase"18.825.720.924.2a-Lactal-bumin69.411.811.318.1DMBA-induced

tumorGalacto

syltransferase1.224.360.890.920.762.55a-Lactal-bummNil2.15NilNilNilNil

mm.6

" nmol of A/-acetyllactosamine formed per mg protein per 30

nmol of lactose formed per mg protein per 30 min.

exogenous hormones. Our data show that estradiol andprolactin are both required for maximal stimulation of a-lactalbumin, provided that the mammary tissue has alreadybeen primed by a combination of estradiol and progesterone to promote cellular proliferation. A combination ofestradiol and prolactin, however, does not seem to offermuch advantage over prolactin alone in the stimulation ofgalactosyltransferase activity (Table 2). The dose ratio ofestradiol to progesterone used in this study was based onthe work of Moon ef al. (25), in which they showed that adose of 1 /Â¿gestradiol and 3 mg progesterone given dailyfor 19 days was optimal for stimulating mammary glanddevelopment equivalent to that of late pregnancy. Dao ef al.(17) also showed that in the rat mammary gland expiantsystem, progesterone, when combined with insulin, prolactin, and estrogen, greatly stimulated DMA synthesis andlobuloalveolar development.

Although not much is known about the regulatory mechanisms in vivo, the induction of both galactosyltransferaseand a-lactalbumin in the in vitro experiments has been welldocumented (39, 42), especially in the mouse mammarygland expiant. In this system prolactin triggers the synthesisof both proteins in cells that have already developed secretory characteristics in response to insulin and hydrocorti-sone. Our study similarly demonstrates the key role ofprolactin in vivo, although in this case the presence ofestrogen is necessary, especially for maximal stimulation ofa-lactalbumin. Estrogen may in some way sensitize themammary tissue to the action of pituitary hormones (37).Sinha ef al. (36) also showed that the effect of prolactin inDMBA-induced mammary tumorigenesis is augmented inthe presence of estrogen. The mechanism of interaction ofthese 2 hormones at the cellular level is unclear at this time.

The inhibitory effect of progesterone on galactosyltransferase and a-lactalbumin levels has been reported earlierby KÃ¼hnef al. (19) and Murphy ef al. (26). They demonstrated that bilateral ovariectomy of late pregnant ratsresulted in an increase in the levels of both proteins thatcould be prevented by progesterone injections subsequentto the operation. Progesterone presumably acts directly onthe mammary tissue since the addition of this steroidhormone in vitro blocks the induction of a-lactalbumin inthe mouse mammary gland organ culture by a lactogeniccomplex of hormones (41). Similarly, high estrogen con

centration in the target tissue caused by direct steroidimplant in the lactating rat mammary gland has been knownto inhibit milk production, estimated by litter weight (48).This study showing the inhibitory effect of a high dose ofestradiol on galactosyltransferase and a-lactalbumin induction provides strong support for this concept. Recent evidence indicates that a combination of estradiol and progesterone inhibits the prolactin-induced increases in RNA andnitrogen content in the mammary gland (11).

We have confined our investigation to the effects ofestradiol and prolactin in the stimulation of galactosyltransferase and a-lactalbumin. In addition to these 2 hormones,the contributions of adrenal corticosteroids (38), placentallactogen (21), and possibly thyroxine (45) to lactose synthesis have been reported. Regulation of lactogenesis/n situ isno doubt a very intricate process and may involve aninterplay of all of these hormones in a coordinate andcomplementary fashion.

The potential of neoplastic mammary cells to synthesizemilk proteins such as a-lactalbumin and casein and theinfluence of hormones on the synthesis of these proteinshave been examined by many investigators. Studies inhuman tumors have thus far been conflicting and very oftendepend on whether determinations were performed onbiopsy samples or in tumors cultured in vitro (8, 16, 24, 29,31, 47). Information on this aspect in animal tumors isprimarily derived from studies involving either the hormone-responsive, transplantable R3230AC mammary adenocarci-noma or the DMBA-induced mammary adenocarcinoma.

In the R3230AC mammary adenocarcinoma system, Hilf(14) reported that tumor fluid aspirated from the neoplasmfollowing 3 weeks of in vivo estradiol treatment containedlactose, fatty acids, and proteins with electrophoretic properties similar to those of casein and whey of rat milk, a-Lactalbumin activity has also been detected in this tumor(22). Nardacci and McGuire (27) demonstrated that caseinmRNA but not a-lactalbumin mRNA was stimulated byexogenous prolactin. Similarly, casein synthesis can alsobe augmented in the presence of insulin, hydrocortisone,and prolactin in the expiant system (43). Using a radioim-munoassay technique Schultz and Ebner (34) showed thatserum a-lactalbumin levels correlated well with the growthof the R3230AC tumors, suggesting that secretory tumorsmay be monitored by serum a-lactalbumin.

Dao ef al. (9) have previously reported that DMBA-induced mammary tumor expiant failed to respond to hormones in the stimulation of casein synthesis. On the otherhand, high-dosage estradiol administration in vivo increases the secretory activity of DMBA-induced mammarytumors (1, 15). Recently, Qasba and Gullino (30) reportedthat DMBA-induced mammary carcinomas contained a-lactalbumin, determined by radioimmunoassay, in quantitiesequal to or less than 10% of the amount found in 3- to 5-daylactating mammary gland. With an enzymatic assay ourresults (Table 3) showed that only 4 of 15 tumors containedmeasurable amounts of a-lactalbumin at a level about 3 to5% of that found in lactating mammary gland. Synthesis ofa-lactalbumin in these 4 tumors could be marginally stimulated by a combination of estradiol and prolactin. Theamount of a-lactalbumin in the tumor reported by Qasbaand Gullino is within the detection limit of our assay

JULY 1978 2081



C. Ip and T. L. Dao

method. Although the radioimmunoassay is a more sensitive technique, this method nonetheless measures biologically active a-lactalbumin activity.

Previous work by Sinha ef a/. (36) has shown that bothestrogen and prolactin are required for the growth ofDMBA-induced mammary tumors. This study demonstratesthat most of the tumors are immune to hormonal stimulation in the synthesis of a-lactalbumin, a protein that isunique to the differentiated mammary cells. Thus, thereseems to be a distinction between the hormonal control ofgrowth and functional differentiation in the DMBA-inducedmammary tumor, and these 2 processes seem to be independent of each other.

There are several reports that indicate that prolactin mayincrease the estrogen receptor level in DMBA-inducedmammary tumors (13, 32, 44) as well as in human breastcancer cells (35). DeSombreef al. (10) have examined 24DMBA-induced mammary tumors and found that essentiallyall of the tumors contained some specific estrogen receptorand that all but 3 contained prolactin receptor. There was,however, no clear-cut correlation between the levels ofthese 2 receptors and the hormone dependency of thetumors as determined by their growth response to ovariec-tomy or hypophysectomy. In view of the evidence thatmaximal a-lactalbumin production is dependent on prolactin and estrogen stimulation and that prolactin may regulatethe level of estrogen receptor, it would therefore be interesting to see if there is any correlation between the capability of the tumor to synthesize a-lactalbumin in response toprolactin and the estrogen-binding capacity of the tumor.The results in Table 3 show that of the 4 tumors with thehighest estrogen-binding capacity, 3 were capable of producing a-lactalbumin. Although it would be premature todraw any conclusion at this point, the data suggest that thedelineation of certain hormone-induced biochemical parameters should be considered in conjunction with receptorlevel to define better the nature of hormone dependency inthe tumor.

As shown in the results in Table 4, the inability of thetumors to synthesize a-lactalbumin, even when the host isunder the influence of endogenous lactogenic hormones inthe course of pregnancy and lactation, suggests that thegene coding for the synthesis of a-lactalbumin is probablynot expressed as a result of malignant transformation. Inthe rats carrying mammary tumors, the host mammarygland retains the ability to lÃ¡clate, although at a reducedcapacity. This experiment, however, fails to show whetherthe impairment is due to some subtle DMBA-induced injuryto the mammary tissue and/or an inadequate galactopoieticstimulation due to the tendency of the dams to reject thepups.

On the basis of the evidence in this study, it can beinferred that only a minority of DMBA-induced mammarytumors retain the potential to manifest functional differentiation. However, the data are still too inconclusive at thisjuncture to show many common characteristics, particularly in relation to the estrogen-binding capacity of thetumor.

ACKNOWLEDGMENTSThe authors are grateful for the technical assistance of Rose Marie VÃ¡rela,

Cassandra Hayes, and Deborah Ketch.

REFERENCES

1. Archer, F. L. Fine Structure of Spontaneous and Estrogen-InducedSecretion in Breast Tumors in the Rat Induced by 7,12-Dimethylbenz[a]anthracene. J. Nati. Cancer Inst., 42: 347-351, 1969.

2. Boylan, E. S.. and Witiiirf, J. L. Specific Estrogen Binding in RatMammary Tumors Induced by 7,12-Dimethylbenz(a)anthracene. CancerRes.,35: 506-511, 1975.

3. Brew, K., Vanaman, T. C., and Hill, R. L. The Role of a-Lactalbumin andthe A Protein in Lactose Synthetase: A Unique Mechanism for theControl of a Biological Reaction. Proc. Nati. Acad. Sei. U. S., 59: 491-497, 1968.

4. Brodbeck, U., DentÃ³n, W. L., Tanahashi, N., and Ebner, K. E. TheIsolation and Identification of the B Protein of Lactose Synthetase as a-Lactalbumin. J. Biol. Chem., 242: 1391-1397, 1967.

5. Brodbeck, U., and Ebner, K. E. Resolution of a Soluble Lactose Synthetase into Two Protein Components and Solubilization of MicrosomalLactose Synthetase. J. BioJ. Chem., 247: 762-764, 1966.

6. Burton, K. A Study of the Conditions and Mechanisms of the Diphenyla-mine Reaction for the Colorimetrie Estimation of DNA. Biochem. J., 62:315-323,1956.

7. Coffey, R. G., and Reithel, F. J. The Lactose Synthetase Particles ofLactating Bovine Mammary Gland. Biochem. J., 109: 169-176, 1968.

8. Daehnfeldt, J. L., and Schulein, M. High Affinity Estradici Receptors andthe Activity of Glucose-6-phosphate Dehydrogenase and Lactose Synthetase in Mammary Carcinoma of Postmenopausal Women. Brit. J.Cancer, 37: 424-428, 1975.

9. Dao, T. L., Sinha, D. K., Christakos, S., and VÃ¡rela, R. BiochemicalCharacterization of Carcinogen-induced Mammary Hyperplastic AlveolarNodule and Tumor in Rat. Cancer Res., 35: 1128-1134, 1975.

10. DeSombre, E. R., Kledzik, G., Marshall, S., and Meites, J. Estrogen andProlactin Receptor Concentrations in Rat Mammary Tumors and Response to Endocrine Ablation. Cancer Res.,36: 354-358, 1976.

11. Ferreri, L. F., and Griffith, D. R. Inhibition of Experimental LactationalMammary Gland Growth in the Rat with Exogenous Estrogen andProgesterone. Proc. Soc. Exptl. Biol. Med., 755: 429-432, 1977.

12. Frantz, W. L., and Turkington, R. W. Formation of Biologically Active125l-Prolactin by Enzymatic Radioiodination. Endocrinology, 97: 1545-1548,1972.

13. Gibson, S. L., and Hilf, R. Influence of Hormonal Alteration of Host onEstrogen-binding Capacity in 7,12-Dimethylbenz(a)anthracene-inducedMammary Tumors. Cancer Res., 36: 3736-3741, 1976.

14. Hilf, R. Milk-Like Fluid in Mammary Adenocarcinoma: BiochemicalCharacterization. Science, 755: 826-827, 1967.

15. Kiang, D.T., and Kennedy, B. J. Combination of Cyclophosphamide andEstrogen Therapy in DMBA-induced Rat Mammary Cancer. Cancer, 28:1202-1210,1971.

16. Kleinberg, D. L. Human a-Lactalbumin: Measurement in Serum and inBreast Cancer Organ Cultures by Radioimmunoassay. Science, 790:276-278. 1975.

17. Koyama, H., Sinha, D. K., and Dao, T. L. Effects of Hormones and 7,12-Dimethylbenz[a]anthracene on Rat Mammary Tissue Grown in OrganCulture. J. Nati. Cancer Inst.,48: 1671-1680, 1972.

18. KÃ¼hn,N. J. Lactogenesis in the Rat. Biochem. J., 706: 743-748, 1968.19. KÃ¼hn,N. J. Progesterone Withdrawal as the Lactogenic Trigger in the

Rat. J. Endocrinol.,44: 39-54, 1969.20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein

Measurement with the Folin Phenol Reagent. J. Biol. Chem., 793: 265-275,1951.

21. Matthies, D. L. Studies of the Luteotrophic and Mammotrophic FactorFound in Trophoblast and Maternal Peripheral Blood of the Rat atMidpregnancy. Anat. Record, 759: 55-68, 1967.

22. McGuire, W. L. Hormonal Stimulation of Lactose Synthetase in Mammary Carcinoma. Science, 765: 1013-1014, 1969.

23. McKenzie, L., Fitzgerald, D. K., and Ebner, K. E. Lactose SynthetaseActivities in Rat and Mouse Mammary Glands. Biochim. Biophys. Acta,230:526-530, 1971.

24. Monaco, M. E., Bronzert, D. A., Tormey, D. C., Waalkes, P., andLippmann, M. E. Casein Production by Human Breast Cancer. CancerRes., 37: 749-754, 1977.

25. Moon, K. C., Griffith, D. R., and Turner, C. W. Normal and ExperimentalGrowth of Rat Mammary Gland. Proc. Soc. Exptl. Biol. Med., 707: 788-790,1959.

26. Murphy, G., Sriyanayagam, A. D., and KÃ¼hn,N. J. Progesterone and theMetabolic Control of the Lactose Biosynthetic Pathway during Lactogenesis in the Rat. Biochem. J., 736: 1105-1116, 1973.

27. Nardacci, N. J., and McGuire, W. L. Casein and a-Lactalbumin Messenger RNA in Experimental Breast Cancer. Cancer Res., 37: 1186-1190,1977.

28. Palmiter, R. D. Hormonal Induction and Regulation of Lactose Synthetase in Mouse Mammary Gland. Biochem. J., 773: 409-417,1969.

29. Pich, A., Bussolati, G., and DiCarlo, F. Production of Casein and thePresence of Estrogen Receptors in Human Breast Cancer. J. Nati.Cancer Inst., 58: 1483-1484, 1977.





30. Qasba, P. K., and Cullino, P. M. a-Lactalbumin Content of Rat MammaryCarcinomas and the Effect of Pituitary Stimulation. Cancer Res., 37:3792-3795. 1977.

31. Rose, H. N., and McGrath, G. M. a-Lactalbumin Production in HumanMammary Carcinoma. Science. 790: 673-675. 1975.

32. Sasaki, G. H., and Leung, B. Prolactin Stimulation of Estrogen Receptorin Vitro in 7,12-Dimethylbenz[a]anthracene-lnduced Mammary Tumor.Res. Commun. Chem. Pathol. Pharmacol.,8: 409-412, 1974.

33. Schneider, S. L., and Dao, T. L. Effect of Ca2' and Salt on Forms of

Estradici Cytoplasmic Receptor in Human Neoplastic Breast Tissue.Cancer Res., 37: 382-387, 1977.

34. Schultz, G. S., and Ebner, K. E. Measurement of a-Lactalbumin inSerum and Mammary Tumors of Rats by Radioimmunoassay. CancerRes., 37: 4482-4488, 1977.

35. Shafie, S., and Brooks, S. C. Effect of Prolactin on Growth and theEstrogen Receptor Level of Human Breast Cancer Cells (MCF-7). CancerRes.,37: 792-799, 1977.

36. Sinha, D., Cooper, D., and Dao, T. L. The Nature of Estrogen andProlactin Effect on Mammary Tumorigenesis. Cancer Res., 33: 411-414,1973.

37. Talwalker, P. K., and Meites, J. Mammary LÃ³bulo-Alveolar GrowthInduced by Anterior Pituitary Hormones in Adreno-Ovariectomized andAdreno-Ovariectomized-Hypophysectomized Rats. Proc. Soc. Exptl.Biol. Med., 107: 880-883,1961.

38. Talwalker, P. K., Nicoli, C. S., and Meites, J. Induction of MammarySecretion in Pregnant Rats and Rabbits by Hydrocortisone Acetate.Endocrinology, 69: 802-808. 1961.

39. Topper, Y. J., and Oka. T. Some Aspects of Mammary Gland Develop

ment in the Mature Mouse. In: B. L. Larson and V. R. Smith (eds.),Lactation, Vol. 1, pp. 327-348. New York: Academic Press, Inc., 1974.

40. Turkington, R. W., Brew, K., Vanaman, T. C., and Hill, R. L. TheHormonal Control of Lactose Synthetase in the Developing MouseMammary Gland. J. Biol. Chem.. 243. 3382-3387, 1968.

41. Turkington, R. W., and Hill, R. L. Lactose Synthetase: ProgesteroneInhibition of the Induction of a-Lactalbumin. Science, 763: 1458-1460,1969.

42. Turkington, R. W., Majumder, G. C., Kadohama, N., Maclndoe, J. H.,and Frantz, W. L. Hormonal Regulation of Gene Expression in MammaryCells. Recent Progr. Hormone Res., 29: 417-455, 1973.

43. Turkington, R. W., and Riddle, M. Expression of Differentiated Functionof Mammary Carcinoma Cells in Vitro. Cancer Res., 30: 127-133, 1970.

44. Vignon. F.. and Rochefort, H. Regulation of Estrogen Receptors inOvarian-Dependent Rat Mammary Tumors. I. Effects of Castration andProlactin. Endocrinology, 98: 722-729, 1976.

45. Vonderhaar, B. K. A Role of Thyroid Hormones in Differentiation ofMouse Mammary Gland in Vitro. Biochem. Biophys. Res. Commun., 67:1219-1225, 1975.

46. Vonderhaar, B., Owens, I. S., and Topper, Y. J. An Early Effect ofProlactin on the Formation of a-Lactalbumin by Mouse MammaryEpithelial Cells. J. Biol. Chem., 248: 467-471, 1973.

47. Woods, K. L., Cove, D. H., and Howell, A. Predictive Classification ofHuman Breast Carcinomas Based on Lactalbumin Synthesis. Lancet, 2:14-16, 1977.

48. Zamora, A. J., Mendoza, A. S., and Ramirez, V. D. Ultrastructure of theMammary Gland in Lactating Rats after Local Implantation of Estrogen.J. Endocrinol.,66: 177-182, 1975.

JULY 1978 2083



1978;38:2077-2083. Cancer Res Clement Ip and Thomas L. Dao Tumor-Lactalbumin Activities in Rat Mammary Gland and Mammary

αEffect of Estradiol and Prolactin on Galactosyltransferase and

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