Vol. 7, 3-11, January 1996 Cell Growth & Differentiation 3

Expression of a Viral Oncoprotein during Mammary GlandDevelopment Alters Cell Fate and Function: Induction ofp53-independent Apoptosis Is Followed by ImpairedMilk Protein Production in Surviving Cells1

Minglin Li, Jiadi Hu, Kathnn Heermeier,Lothar Hennighausen, and Priscilla A. Furth2Division of Infectious Diseases, Department of Medicine, University of

Maryland Medical School and the Baltimore Veterans Affairs Medical

Center, Baltimore, Maryland 21201 [M. L, J. H., P. A. F.], andLaboratory of Biochemistry and Metabolism, National Institutes ofDiabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland

20892 [K. H., L. H.]

AbstractThe disruption of cell cycle regulation is associatedwith developmental abnormalities and tumorigenesis.The SV4O large T antigen (Tag) interferes with cellcycle control by interacting with the pRb family andp53. Mice carrying a transgene composed of thewhey acidic protein (WAP) gene promoter and theTag coding sequence express Tag during pregnancyand are unable to nurse their young. Tag expressioninduced apoptosis in mammary epithelial cells duringlate pregnancy. At least 5% of mammary epithelialcells were undergoing apoptosis at any one time. Incontrast, less than 0.2% of mammary epithelial cellsin nontransgenic littermates was undergoingapoptosis. Apoptosis in Tag mice was associatedwith increased steady-state RNA levels of bax andbcl-xL+S, with a relative increase in bcI-x�expression. Since p53 was sequestered by Tag, it islikely that p53-independent mechanisms precipitatedapoptosis. The Tag-expressing mammary alveolarcells that did not undergo apoptosis continued todifferentiate through late pregnancy, as measured bythe sequential activation of milk protein geneexpression. However, milk protein production,processing, and secretion was impaired, resulting inlactation failure.

Received 6/19/95; revised 10/3/95; accepted 10/5/95.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 1 8 U.S.C. Section 1 734 solely to mdi-

cate this fact.I This work was supported in part by a research contract from Galagen(Arden Hills, MN; to P. A. F.) and Grant 01-067482532 from the VeteransAdministration Research Advisory Group (to P. A. F.).2 To whom requests for reprints should be addressed, at Department ofMedicine, Room 5D-136, Baltimore VA Medical Center, University ofMaryland Medical School, 10 North Greene Street, Baltimore, MD 21201.Phone: (410) 605-7181; Fax: (410) 605-7914; E-mail: furth@ncifcrf.gov.

IntroductionThe pRb3 family and p53 have critical functions in regulatingthe cell cycle. In transgenic mice, expression of the SV4Olarge Tag and the papillomavirus E6 and E7 proteins can leadto developmental defects and cancer by disrupting cell cyclecontrol through interactions with pRb and p53 (1-4). Weused mice carrying a transgene composed of the WAP pro-moter and the Tag coding sequence (5) to identify cellularpathways disrupted through the expression of this viral on-coprotein in the developing mammary gland.

Mammary gland development consists of well characterizedsteps that culminate in lactation (6-9). Developmental abnor-malities at specific stages can be recognized through the ex-amination of mammary gland structure and the evaluation ofcellular differentiation. The function of the gland can be as-sessed by the presence or absence of a successful lactation. Invirgin mice, the mammary parenchyma is composed of anorganized system of ducts within the mammary fat pad. Themajor sites of growth are at the terminal end buds. With eachestrous cycle, the lateral end buds differentiate and subdivideprogressively. Extensive development mediated by lactogenichormones begins with pregnancy and is completed at the onsetof lactation. A rapid increase in the number and size of alveolioccurring during the second half of pregnancy results in devel-opment of fully differentiated and functional secretory lobules.Differentiation of mammary epithelial cells and formation ofalveolar structures during pregnancy leads to the sequentialexpression of milk protein genes (7), followed by milk proteinsecretion and lactation. Lactation is maintained as long as thedams are suckled. After weaning, the mammaty gland invo-lutes, and the entire lobulo-alveolar compartment collapsesthrough PCD. This results in a ductal network resembling that ofa mature virgin. The mammary gland can serve as a paradigm

to investigate how loss of cell cycle regulation, mediated by aviral oncoprotein, affects both organ development and function.

Female mice carrying a WAP-Tag transgene specificallyexpress Tag in mammary tissue beginning around day 13 ofpregnancy (5), but tumor development does not occur untilafter three to five pregnancies. These mice are unable tonurse their young, starting with the first pregnancy. Thissuggests that expression of a viral oncoprotein leads tofunctional defects in mammary epithelial cells prior to malig-nant transformation.

3 The abbreviations used are: pRb, retinoblastoma protein; Tag, SV4Olarge T antigen; WAP, whey acidic protein; PCD, programmed cell death;nt, nucleotide; TGF�31 , transforming growth factor �31 ; MDGI, mammary-derived growth inhibitor; RT-PCR, reverse transcriptase PCR.

4 5V40 Tag Alters Mammary Cell Fate and Function

Fig. 1. Mammary glands from

Tag mice exhibit decreased alve-olar density as compared to con-trol mice. Whole-mount analysisof mammary glands from Tag (Aand C) and control (B and D)mice. The inguinal glands of day16 pregnant mice were mountedon a glass slide, fixed, andstained. Similar results were

seen in glands from late preg-nancy and on the day of parturi-tion. A and B show the entiregland; C and D show the sameglands at increased magnifica-tion. LN, lymph node.

In this study, we demonstrate that the presence of Tag indifferentiating mammary epithelial cells altered both cell fate

and function. Tag synthesis induced p53-independent ap-optosis in mammary epithelial cells during late pregnancy.This was probably mediated by changes in bax and bcl-xL#{247}Sexpression. However, only a subset of Tag-expressing cellsunderwent apoptosis, and alveolar structures were not de-

stroyed. Tag-expressing cells that did not undergo apoptosiscontinued to differentiate throughout late pregnancy andexpressed major milk protein RNAs. However, milk proteinproduction and secretion were impaired in these cells, re-suiting in lactation failure.

ResultsTag Mice Cannot Nurse Their Offspring. Tag was de-tected in alveolar cells at approximately day 1 3 of the firstpregnancy. Although nontransgenic females nursed all oftheir litters, Tag mice were unable to feed their offspring,beginning with the first litter. Pups delivered to transgenicfemales were healthy initially and attempted to suckle.However, no milk was observed in their stomachs, and100% of the offspring died within 48 h. In cross-fosteringexperiments, 76% of offspring born to Tag mice could berescued by fostering them to a nontransgenic female.Conversely, 1 00% of offspring born to nontransgenic micedied after being fostered to Tag mice (data not shown). Toinvestigate why Tag mice cannot lactate at the time ofdelivery, studies were focused on the developmental stateof the gland just prior to delivery. In normal mice, milkproduction and secretion into the alveoli begins a few daysprior to delivery. Gestation in the Tag mice and controlswas 20 days. Mice were evaluated during the first preg-nancy to identify events that occur shortly after Tagexpression.

Tag-induced, p53-independent Apoptosis Was Associ-ated with Increased Steady-State Levels of bax andbcl-xL+s RNA. Whole-mount analyses of mammary tissuefrom Tag mice revealed decreased alveolar density, starting

at day 16 of pregnancy through parturition (Fig. 1). Histolog-cal analyses demonstrated that the average alveolar

circumference of Tag mice was smaller than that of controlmice (Fig. 2).

In situ detection of apoptotic cells demonstrated that ap-

proximately 5% of mammary epithelial cells were activelyundergoing apoptosis in Tag mice at day 18 to day 19 ofpregnancy (Table 1 ; Fig. 2, G and H). In contrast, less than0.2% of cells underwent apoptosis in mammary glands ofnontransgenic pregnant mice. Tag expression was detectedin the majority of mammary epithelial cells (Fig. 2, D and E).

Immunohistochemical staining localized p53 to the nucleusof epithelial cells of transgenic mice but was undetected innontransgenic mice (data not shown; Ref. 5). These resultssuggest that the apoptosis induced by Tag expression dur-ing pregnancy was mediated by p53-independent mecha-nisms. To identify which apoptosis pathway genes weretranscriptionally activated following Tag expression, we per-

formed Northern blot analyses of steady-state levels of bax,bcl-xL±s, p53, and bcl-2 on RNA extracted from mammaryglands of late pregnant mice. Steady-state levels of bax (Fig.

3C) and bcl-xL#{247}s (Fig. 3A) RNA were increased approxi-mately 5- and 2-fold, respectively, as compared to nontrans-

genic controls. To distinguish between bcl-xL and bcl-x�

RNA, a RT-PCR analysis was performed with primers whichsimultaneously amplified both forms, followed by a hybrid-

ization with oligonucleotides that distinguished between the

two forms. Both bcl-xL and bcl-x� RNA levels were increased

in Tag transgenic mice, but a relatively greater increase in

bcl-x� RNA levels was observed (Fig. 3B). Bcl-xL RNA re-

Fig. 2. Tag-induced apoptosis in mammary epithelial cells during pregnancy. Histological analysis of mammary tissue from day 18 pregnant Tag (A, B,D, E, G, and H) and control (C, F, and I) mice. Tissue was sectioned and stained with H&E (A, B, and C). Fat pad, alveoli, lumen, and secretions are indicated.A: arrow, an apoptotic cell. lmmunohistochemical analysis for the presence of Tag was performed (0, E, and F). D, solid arrow, a Tag-expressing cell. Note:the majority of nuclei in the Tag mice are positive for Tag. In situ detection of apoptosis was performed (G, H, and I). G: arrows, apoptotic cells. Cellsundergoing apoptosis appear brown.

Cell Growth & Differentiation 5

Table 1 Percentage of cells undergoing apoptosis in sections of mammary gland tissue from control (C) and Tag (1) mice

Each mouse no. represents ma mmary tissue taken from a different mouse. A poptotic cells we re identified usin g the Apotag kit (0 ncor, Gaithersburg, MD).

Control Tag

Mouse flO.aApoptotic

cellsTotal cells

counted#{176}“#{176}apoptotic cells Mouse no.

Apoptoticcells

Total cellscounted

% apoptotic cells

1 2 1035 0.19 5 48 1064 4.52 2 1040 0.19 6 43 1079 4.0

3 2 1039 0.19 7 60 1110 5.4

4 2 1042 0.19 8 64 1046 6.1

Mean 2 1039 0.19 Mean 54 1075 5.0

a Mouse 1: C, Pregnancy (P) Day (D) 17. Mouse 2: C, PD 18. Mouse 3: C, PD 18. Mouse 4: C, PD 19. Mouse 5: T, PD 17. Mouse 6: T, PD 18. Mouse 7:

T, PD 18. Mouse 8: T, PD 19.

mained the predominant form in both transgenic and control

mice and was at least 5- to 1 0-fold more abundant than

bcl-x�. No p53 or bcl-2 expression was detected by Northernblot analyses (data not shown). The increased levels of bax

RNA in the Tag mice translated into increased levels of baxprotein (Fig. 3D).

Expression of bcl-xL±S and bax was also evaluated in

mammary gland tumors from Tag mice. This analysis was

performed to determine if the changes observed during

pregnancy persisted and because changes in expression

patterns of apoptosis pathway genes can be associated with

tumor progression (10). Both bcl-xL±S and bax RNA weredetected in tumor specimens (Fig. 3 and data not shown).The bcl-xL:bcl-xS RNA ratio was similar to that found in the

mammary glands from late pregnant Tag mice. These results

indicate that changes in the ratio of bcl-xL to bcl-x� expres-

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6 SV4O Tag Alters Mammary Cell Fate and Function

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Fig. 3. Bax and bcl-xL±S expression increased in Tag mice coincident with the appearance of PCD. A, Northern blot analysis of steady-state RNA levelsof bcl-xL+S at late pregnancy. RNA from control mice, Tag (WAP-Tag) mice, and two Tag-induced mammary gland tumors (Tumor, Lanes 9 and 10) wereanalyzed. Each lane represents a mammary gland or tumor taken from a different mouse. Lane 1, day 17 of pregnancy; Lane 2, day 18; Lane 3, day 18;Lane 4, day 19; Lane 5, day 17; Lane 6, day 18; Lane 7, day 18; Lane 8, day 19. Ethidium bromide staining of 285 and 185 RNA was used as a loadingstandard. B, Southem blot analysis of bcl-xL- and bcl-x�-specific PCR products following RT-PCR. The same samples analyzed in A were used in theRT-PCR assay. C, Northem blot analysis of steady-state bax RNA levels at late pregnancy. A subset of the same samples analyzed in A were examined.Lane 1, day 18 of pregnancy; Lane 2, day 19; Lane 3, day 18; Lane 4, day 19. 0, Western blot analysis of steady-state levels of bax protein at late pregnancyand in tumor tissue. Each lane represents a mammary gland or tumor specimen (Lane 7) taken from a different mouse. Lane 1, day 17 of pregnancy; Lane2, day 18; Lane 3, day 18; Lane 4, day 19; Lane 5, day 18; Lane 6, day 18. Coomassie blue staining of the protein gel demonstrated equal loading of allsamples.

sion were correlated with Tag expression. Elevated concen-trations of bax proteins were also detected in tumor speci-mens (Fig. 3D).

Epithelial Cells Expressing Tag Differentiate ThroughLate Pregnancy as Measured by the Sequential Activa-tion of Milk Protein Genes, but Milk Protein ProductionWas Impaired. Histological examination of mammary tissuefrom day 16 of pregnancy through parturition demonstrateda dramatic reduction in the number of fat globules present inthe alveolar cells. To assess the differentiation state of the

Tag-expressing cells, Northern blot analyses were used toevaluate expression of the differentiation-specific milk pro-tein genes. The milk protein genes f3-casein, WAP, anda-lactalbumin were expressed from late pregnancy throughthe day of delivery (Fig. 4; Ref. 11). Milk protein production

was evaluated from day 17 of pregnancy through parturition.Mammary glands obtained from Tag and control mice duringpregnancy or within 24 h of delivery revealed differences inmilk accumulation upon visual examination. When sectionedin half, milk readily leaked from glands of nontransgenic mice

but not Tag mice. The presence of milk proteins was evalu-

ated by immunohistochemistry and Western blot analysisusing antibodies directed against either mouse WAP or totalmouse milk proteins. Milk proteins were abundant in bothmammary epithelial cells and the alveolar lumens of controlmice (Fig. 5, C and D). In contrast, very little milk protein wasdetected in either epithelial cells or alveolar lumens in Tagmice using immunohistochemistry (Fig. 5, A and B). Westernblot analysis demonstrated decreased levels of WAP proteinin Tag mice and reduced posttranslational processing (Fig.

5E). Both WAP precursor and mature WAP were found inmammary tissue of late pregnant control mice. In contrast,

Tag mice demonstrated only a single WAP precursor bandconsistent with failure of cleavage of the signal peptide (12).

Differential Expression of the Tumor Marker GeneWDNMI in Tag Mice. Northern blot analyses were used to

determine if there was evidence of differential expression of

other developmentally regulated genes. Three additional genes,the expression of which normally increases during pregnancy,were examined: WDNM1, TGFj31, and MDGI. WDNM1 is a

developmentally regulated gene the expression of which isreduced in c-myc and neu but not ras and mt transformed

mammary cells lines (1 3). TGFI31 expression normally increases

during late pregnancy and falls at the time of delivery (14).Expression of MDGI normally increases during differentiation ofmammary epithelial cells (15). Only expression of WDNM1 was

reduced in mammary tissue of Tag transgenic mice during thefirst pregnancy (Fig. 6 and data not shown).

DiscussionThis study demonstrates that expression of a viral onco-

protein in developing mammary gland alters cell fate andfunction (Fig. 7). Shortly after Tag synthesis commenced

during pregnancy, mammary epithelial cells began to un-

dergo PCD. At the same time, cells which expressed Tagbut did not undergo apoptosis were unable to produce

and secrete milk proteins. This occurred despite the pres-ence of sufficient steady-state levels of the correspondingRNAs.

Tag

Cell Growth & Differentiation 7Control WAP-Tag

1234 5678

WAP

Fig. 4. Milk protein RNA expression in control and Tag mice. Northernblot analysis of steady-state RNA levels of �3-casein, WAP, a-lactalbumin,and Tag at late pregnancy in control and Tag mice. Each lane representsa mammary gland taken from a different mouse. Lane 1, day 1 7 ofpregnancy; Lane 2, day 1 8; Lane 3, day 18; Lane 4, day 1 9; Lane 5, day17; Lane 6, day 18; Lane 7, day 18; Lane 8, day 19. Ethidium bromidestaining of 28S and 185 RNA was used as a loading standard.

Tag-induced Apoptosis in Mammary Epithelial Cellsduring Pregnancy. The appearance of large numbers of ap-

optotic cells in the developing mammary gland ofTag mice was

distinctly abnormal. When the pRb-binding portion of Tag is

expressed in the lens or choroid plexus (1-4), it induces pri-manly p53-dependent apoptosis. Since both pRb- and p53-

binding domains of Tag were expressed in mammary tissue

coincident with PCD, we suggest that p53-independent apop-

tosis was activated by expression of Tag in the mammary

gland. This is consistent with the concept that p53-independent

apoptosis is important in normal mammary gland development

and involution. In support of this hypothesis, we performed a

related study that demonstrated that mammary gland involution

does not require functional p53 (11).Different cell types or different physiological conditions

may have different capacities to activate either p53-depen-

dent or p53-independent pathways (1 6, 1 7). This could be

related to developmental needs. For instance, the lens and

choroid plexus develop just once, while mammary glanddevelopment repeats with each pregnancy. The lens andchoroid plexus do not normally undergo extensive apoptosis

during life, while the mammary gland involutes through PCD

following each lactation. These results raise the possibility

that there are distinct roles for p53-independent apoptosis

and p53-dependent apoptosis during mammary gland tu-

morigenesis. The marked induction of apoptosis by Tag dur-

ing pregnancy infers that p53-independent apoptosis could

play a protective role in early steps of Tag-induced tumori-

genesis. However, since resistance to p53-dependent apop-

tosis is associated with tumor progression in the choroid

plexus (4), it is possible that development of resistance to

p53-independent apoptosis pathways contributes to pro-

gression of breast cancer in these mice.

Members of the bax and bcl-x family of genes mediate

apoptosis by forming homo- and heterodimers (1 8). Therelative levels of each protein in an individual cell may de-

termine whether or not a cell will undergo apoptosis (19).

Steady-state levels of both bax, which promotes cell death

(20), and bcl-xL±S RNA were increased in the Tag mice

coincident with the appearance of PCD. Bcl-x can be tran-

scribed and processed into two different RNA forms with

opposing functions (21 , 22). Bcl-xL is the most abundantform and inhibits PCD. Bcl-x� is expressed at lower levels

and accelerates cell death in transfection studies in vitro (21).

Because of its lower expression levels, the role of bcl-x� ininducing PCD in vivo has not been obvious (22). Similar toprevious studies, we found that bcl-xL was the predominant

RNA form expressed. However, a relative increase in bcl-x�expression as compared to bcl-xL expression was detectedcoincident with Tag-induced apoptosis. Since a similar in-

crease in bcl-x� expression has been described during nor-

mal involution of the mammary gland (11),� increased splic-

ing efficiency toward the short form may contribute to the

appearance of apoptosis in the mammary gland. Although

p53 can activate transcription of the bax gene (23), thesestudies suggest that activation does not require p53.

Expression of Tag during Pregnancy Led to ImpairedMilk Protein Production and Failure of Lactation. Tagsynthesis in mammary epithelial cells did not destroy alveolar

structures but resulted in impaired milk protein production

and secretion, despite sufficient steady-state amounts of

milk protein mRNAs. Intraluminal milk protein secretion was

sharply reduced in the presence of Tag. Western blot anal-

yses indicated that the milk protein present was not pro-cessed and secreted properly. Some WAP precursor wasdetected on Western blots but not on immunocytochemistry,

suggesting that unprocessed WAP, which is not secreted

into the endoplasmic reticulum and lumen, is not efficiently

recognized within tissue sections. These results further dem-onstrated that expression of a viral oncoprotein impaired

cellular function prior to evidence of malignant transforma-

tion. It is not known whether the production and processing

of nonmilk proteins were also affected.Milk protein production in Tag mice may be inhibited by

either direct or indirect mechanisms. It does not appear that

4 K. Heermeier and L Hennighausen. Bax and bcl-x� are induced at theonset of apoptosis in involuting mammary epithelial cells, submitted forpublication.

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8 SV4O Tag Alters Mammary Cell Fate and Function

Fig. 6. Differential expression ofWDNM1 RNA in Tag mice. Northern blotanalysis of steady-state RNA levels of WDNM1 and TGFf31 at late preg-nancy in control (Lanes 1-4) and Tag (Lanes 5-8) mice. Each lane rep-resents a mammary gland taken from a different mouse. Lane 1, day 1 7 ofpregnancy; Lane 2, day 18; Lane 3, day 18; Lane 4, day 19; Lane 5, day17; Lane 6, day 18; Lane 7, day 18; Lane 8, day 19. Ethidium bromidestaining of 285 and 185 RNA was used as a loading control and isillustrated in Fig. 4.

direct apoptotic destruction of the gland is responsible sincealveolar structures and milk protein mRNAs are present

throughout late pregnancy and parturition. In Tag mice,mammary gland involution and lobulo-alveolar collapse withreduced levels of milk protein mRNAs does not occur untilafter parturition and lactation failure (1 1). There are severalmechanisms through which Tag could act to inhibit milk

Fig. 5. lmmunoNstochemic� (4-0) andWestern blot (E) analyses of WAP and to-tal milk proteins in Tag (A and B) andcontrol (C and D) mice. Immunohisto-chemical analyses for the presence ofWAP (A and C) and total milk protein (Band D) was performed. E, Western blotanalysis of steady-state levels of WAPprotein at late pregnancy in control (Lanes1 and 2) and Tag (Lanes 3-5) mice. Each

lane represents a mammary gland takenfrom a different mouse. Lane 1, day 1 8 ofpregnancy; Lane 2, day 18; Lane 3, day 18;Lane 4, day 19; Lane 5, day 18. The upperband (open arrow) points to the WAP pre-cursor, and the lower band (solid arrow)points to mature WAP. Coomassie Bluestaining of the protein gel revealed equalloading of all samples.

production. Tag synthesis might affect milk protein produc-tion by association with pRb family members and dysregu-lation of cell cycle control (1-4). In muscle cells, functional

pRb is required for terminal differentiation, which is coupledwith cessation of cell divisions (24). Tag could inhibit milk

protein production by interfering with the ability of mammary

epithelial cells to exit from the cell cycle and undergo termi-

nal differentiation. In normal mammary tissue, epithelial cell

proliferation falls during late pregnancy, coincident with theonset of milk protein synthesis. pRb or a related family mem-ber could be required for terminal differentiation and exitfrom the cell cycle of mammary epithelial cells. Exit from the

cell cycle could be required for proper and abundant milkprotein synthesis. This hypothesis can be tested by gener-

ating transgenic mice that carry either truncated Tag or mu-

tant Tag constructs (2, 4). Conditional control of Tag expres-

sion using the tet-responsive system could be used to

establish if the effect of Tag on lactation was reversible (25,26). It appears less likely that interruption of p53 function byTag was responsible for the inability to produce milk proteinssince p53 -I- mice lactate normally. It is, however, possiblethat these mice lactate in the absence of p53 due to corn-pensation from another gene product selected for duringembryonic development.

The presence of Tag in mammary epithelial cells couldblock the required growth-suppressive effects of TGFI3 pro-

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Cell Growth & Differentiation 9

Fig. 7. Model illustrating theeffect of disrupting the cell cy-cle by expression of Tag duringmammary gland development.Mammary gland stem cells giverise to alveolar structures dur-ing pregnancy. In Tag mice,expression of the viral oncopro-tein during pregnancy is fol-lowed by induction of p53-independent PCD in alveolarcells. Surviving cells expressnormal amounts of milk proteinRNA but do not produce milkproteins. Expression of thetumor marker gene WDNM1is down-regulated. In controlmice, synthesis and secretionof milk proteins begins in latepregnancy. When pups beginto nurse, lactation starts, andthe diameter of the alveolar lu-men increases. In Tag mice, nomilk protein production occurs,lactation cannot be estab-lished, and the lumen diameterremains small.

teins during pregnancy. TGF�1 and TGFI33 are synthesizedat high levels in the mammary gland during pregnancy anddecrease at parturition (14). Their growth-inhibitory proper-ties may be necessary for alveolar cells to proceed intoterminal differentiation at late pregnancy. TGFI3 family mem-bers block cell proliferation, in part, by maintaining pRb in anactive hypophosphorylated form. Tag binding to pRb wouldinterrupt this effect on mammary epithelial cells (27, 28). Inaddition, the association of Tag with pRb would result inrelease of E2F. Elevated levels of E2F1 expression can blockgrowth suppressive effects of TGFf31 in cell culture (29).

It is possible that Tag synthesis affected fat-related geneexpression or synthesis since the mammary epithelial cells ofTag mice exhibit a marked reduction in the number of fatglobules. Impairment of fat production similar to that seen formilk protein production would contribute to poor milk pro-duction. Finally, since Tag can interact with other cellularproteins than pRb and p53, it is possible that one of theseinteractions also contributed to the impairment of milk pro-duction (30-34).

In summary, expression of Tag in mammary epithelial cellsduring pregnancy altered mammary epithelial cell fate andfunction. The induction of apoptosis in the presence of Tagillustrates the important role of p53-independent apoptosispathways in the mammary gland. The failure of milk proteinproduction following Tag expression indicates that factorsbeyond transcriptional control of milk protein gene expres-sion are required for efficient milk production and lactation.

Materials and MethodsTransgenic Mice, Analysis of NursIng Behavior, Cross-Fostering,Mammary Gland BIopsies, and Tumor Specimens. Transgenic mice

carrying a WAP-Tag hybrid gene were a gift from Adolf Graessmann (Freie

Universit#{228}t, Berlin, Germany; Ref. 5). The WAP-Tag hybrid gene consists

of a 1600-bp WAPgene promoter(BgllI-Kpnl fragment) linked to the 5V40

early coding region (Bglll-BamHI fragment) containing the coding se-quence for both large Tag and small Tag. Progeny mice containing theWAP-Tag transgene were screened using the PCR. The transgene was

identified using primers corresponding to the WAP promoter from nude-

otide nt -88 to nt -68 (5’ TAG AGC TGT GCC AGC CTC TTC 3’) and Tagsequences from nt -4950 to nt -4931 (5’ CAG MG CCT CCA AAG TCAGG 3’).

Both transgenic and nontransgenic littermates were observed andanalyzed during pregnancy and after parturition. Pups bom to these micewere observed after delivery and assessed for suckling behavior and the

presence of milk in the stomachs. To cross-foster pups, matings oftransgenic and nontransgenic females were synchronized, and the off-

spring of transgenic and nontransgenic mice were exchanged within 24 h

of delivery.Mammary gland biopsies were performed between days 16 and 19 of

the first pregnancy. Mice were anesthetized using 0.7 ml of 0.175%avertin i.p. Under sterile conditions, the inguinal mammary gland from

either the right or left side was exposed and removed. The mice recoveredfrom the anesthesia uneventfully and went on to deliver at the normal time.Tumor specimens used in the present study were obtained from two

different mice harvested by biopsy of the affected mammary gland. Thesetumors first appeared after three pregnancies.

Mammary Gland Whole-Mount Preparations. Each whole mam-mary gland specimen was spread on a glass slide and fixed in Camoy’s

solution (100% ethanol:chloroform:glacial acetic acid, 6:3:1) for 60 mm atroom temperature. Following fixation, the glands were washed with 70%

ethanol for 15 mm, followed by a wash with distilled water for 5 mm. Thestaining of the glands was performed in carmine alum (1 g carmine; Sigma

Chemical Co., St. Louis, MO) and 2.5 g aluminum potassium sulfate(Sigma) in 500 ml water) at 4#{176}Covemlght. The tissues were then dehy-drated and mounted on glass slides using routine methods.

HIstologIcal Examination and lmmunohlstochemlstry. Mammarygland specimens were fixed In 1 0% neutral formalin solution and embed-ded in paraffin using routine methods. Five-p.m tissue sections wereprepared using routine methods for hematoxylin and eosin staining andfor the detection of Tag protein, WAP, and total milk protein. Tag proteinwas detected using the monoclonal antibody Pab 1 01 (Santa Cruz Bio-technology, Inc., Santa Cruz, CA). Tissue sections were inffially treated

10 SV4O Tag Afters Mammary Cell Fate and Function

wfth pepsin (2-10 �g/ml in 0.01 N HCI buffer) for 15 mm at room temper-

ature and quenched with 0.03% H202 in PBS for 30 mm at room tem-perature. After treatment with normal horse serum for 30 mm at roomtemperature, the specimens were incubated for 1 h with a 1 :1000 dilutionof PablOl, followed by an incubation for 1 h with biotinylated horse

antimouse lgG at a I :400-500 dilution (Vectastain ABC kft; Vector, L.ab-oratories, Inc., Burlingame, CA). The color reaction was performed with

0.05% 3,3’-dimethylaminoazobenzene (Sigma) and 0.01 % H202. Sec-

tions were counterstained with hematoxylin. WAP was detected using ananti-WAP polyclonal antibody raised in rabbits (35). Tissue sections weretreated with pepsin, followed by quenching as described above. After

blocking with normal goat serum for 1 h at room temperature, the spec-imens were incubated for 1 h with a 1 :200-400 dilution of the primaryantibody, followed by incubation with biotinylated goat antirabbit IgG(H+L) at a 1 :400 dilution. Total milk protein was detected using RAM/TM(Nordic Immunology, Tilburg, the Netherlands) as a primary antibody

following the same protocol used to detect WAP.Detection of Apoptotic Cell Death in lissue Sections. Mammary

gland specimens werefixed and embedded; then 5-jzm tissue sections wereprepared as described above. Apoptotic cell nuclei were identified using the

Apotag kit (Oncor, Gafthersburg, MD). Sections were initially treated with 20�4ml proteinase K in PBS for 15 mm at room temperature, quenched by

0.003% H202 for 30 mm at room temperature, equilibrated with buffer,incubated with TdT for 20-40 mm at room temperature, washed with wash

stop bufferfor30 mm at37#{176}C,and incubated with anti-digoxigenin for30 mm

at room temperature. Color was developed using 0.05% 3,3’-dimethylami-noazobenzene 0.01% H202 diluted in 0.1 M Tris-HCI (pH 7.5) and counter-stained with meth�4 green. Sections were viewed at x440, and the percent-age of cells with apoptotic cell nuclei was determined. Over 1000 caDs werecounted for each sample. Samples from four mice carrying the WAP-Tagtransgene were compared to samples from four control mice at the same

stage of late pregnancy.

Isolation oflotal RNA, Northern Blot Analysis, and RT-PCR Assay.Total ANA was isolated from indMdual mammary glands using acid-guani-

dinium thkcyanate-phenol-chbrcform extraction (36), and the RNA wasquantitated on a specfrophotometer. For Northern blot analysis, 20 �g ofeach sampiewerefractionated on aformaldehydeagarosegel, transferred toa nylon membrane, and fixed on the membrane by IN irradiation. Gene

expression was detected by hybridization of the membrane overnight withindMdual random primer-labeled probes. Gene expression was quantitatedusing a radloanalytical imaging system (AMBIS, Inc., San Diego, CA). The

following 32P-Iabeled probes were used: WAP (36), �3-casein (36), a-lactai-bumin (7), WDNM1 (7), MDGI (15�, Tag (nt 3808-4826); bd-x1� (mRNA nt

110-394) and box (mRNA nt 138-389). Autoradiograph exposure times:�3-casein, 3 h; WAP, 3 h; a-lactalbumin, 8 h; Tag, 24 h; bcl-x��� 7 days; bax,3 days; WDNM1, 2 h; TGFf31, 7 days. The approximate relative amounts of

bdl-xL and bcl-x� mRNA were measured using RT-PCR assay. Forthe assaypresented In Fig 3, 1 � of each sample was reverse transcribed, and 25

cycles of PCR were used. The signal Obtained was proporbonal to the RNAinput and numberof PCR cycles. The cDNAfor bol-x� was amplffied usinga pair of primers that ampilfy the nucleotide sequence cont�ning the region

ditterent�t s�d in the bcl-x, and bcl-x� mRNAs. The 5’ primer used

correspondsto bcl-x mRNA nt 466-488(5’-GCG CGG GAG GTG AlT CCCATG GC-3’)and the 3’ primer used corresponds to bd-x mRNA nt 891-870(5’-CAT 6CC CGT CAG G.M CCA GCG G-3’). The PCR products werefractionated on a 1.2% agarose gel, transferred to a nylon membrane, and

fixed on the membrane by UV irrad�tion. Expression of bcl-x� was identifiedby hybrithing the membrane overnight with an oligonucleotide specific for

thesplicesitecontained withinthe426-bpbcl-x�PCR product(5’-GGC GGGGCA CTG TGC GTG GMAGC G-3’). Expression of bcl-x� was identified byhybridizing the membrane overnight with an oligonucleotide specIfic for thesplice site contained within the 237-bp bcl-x5 product (5’-CAG AGC liTGAG CAG GACACT1TrGTG G-3’). Autoradiograph exposuretimes: bcI-x�,2 h; bcl-x9, 16 h.

Western Blot Analysis. Proteins were extracted from frozen mam-mary tissue using RIPA buffer (PBS, 1 % NP4O, 0.5% sodium deoxy-

cholate, and 0.1 % 5DS) with protease inhibitors phenylmethylsulfonylfluoride, aprotinin, and sodium orthovanadate. Twenty �g of protein from

each sample were fractionated on a SD5-14% polyacrylamide gel. Pro-teins were transferred onto NOVEX polyvinylidene difluoride membranesusing NOVEX Western Transfer Apparatus. After transfer and blockingwith buffer [5% nonfat milk, 10 m� Tris (pH 7.5), 100 m� NaCI, and 0.1%

Tween 20] for 1 h at room temperature, the membranes were exposed toeither a 1 :400 dilution of rabbit anti-WAP polydlonal antibody(35) or rabbitanti-bax (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature,

followed by exposure to a 1 :4000 dilution of peroxidase conjugate goatantirabbit lgG polydlonal antibody (Sigma) for 1 h at room temperature.The proteins were visualized using the ECL Western blotting protocol(Arnersham, Mington Heights, IL).

AcknowledgmentsWe thank Gertraud Robinson, Gilbert Smith, David Kerr, and Robert Wall

for helpful discussion and Albert Lewis for technical help.

References1 . Pan, H., and Griep, A. E. Aftered cell cycle regulation in the lens of

HPV-1 6 E6 or E7 transgenic mice: implications for tumor suppressor genefunction in development. Genes & Dev., 8: 1285-1299, 1994.

2. Fromm, L, Shawlot, W., Gunning, K., Butel, S. S., and Overbeek, P. A.The retinoblastoma protein-binding region of simian virus 40 large Tantigen alters cell cycle regulation in lenses of transgenic mice. Mol. Cell.Biol., 14: 6743-6754, 1994.

3. Bryce, D. M., Uu, Q., Khoo, W., Tsui, L-C., and Brutman, M. L Pro-gressive and regressive fate of lens tumors correlates with subtle differ-

ences in transgene expression in a-crystallin -SV4O T antigen transgenic

mice. Oncogene, 8: 161 1-1620, 1993.

4. Symonds, H., KraII, L, Remington, L, Saenz-Robles, M., Lowe, S.,

Jacks, T., and Van Dyke, T. p53-dependent apoptosis suppresses tumorgrowth and progression in vivo. Cell, 78: 703-71 1 , 1994.

5. Tzeng, V-J., GuhI, E., Graessmann, M., and Graessmann, A. Breastcancer formation in transgenic animals induced by the whey acidic proteinSV4O T antigen (WAP-SV-T) hybrid gene. Oncogene, 8: 1965-1971 , 1993.

6. Topper, Y. J., and Freeman, C. S. Multiple hormone interactions in thedevelopmental biology of the mammary gland. Physiol. Rev., 60:1049-1056, 1980.

7. Robinson, G., McKnight, R. A,, Smith, G. H., and Hennighausen, L

Mammary epithelial cells undergo secretory differentiation in cycling vir-gins but require pregnancy for the establishment of terminal differentia-

tion. Development (Camb.), 121: 2079-2090, 1995.

8. Russo, I. H., Tewari, M., and Russo, J. Morphology and developmentof the rat mammary gland. In: T. C. Jones, V. Mohr, and R. D. Hunt (eds.),Integument and Mammary Glands, pp. 233-252. Berlin: Springer-Verlag,

1989.

9. Daniel, C. W., and Silberstein, G. B. The mammary gland: develop-ment, regulation and function. New York: Plenum Publishing Corp., 1987.

10. Korsmeyer, S. J. Bcl-2 initiates a new category of oncogenes: regu-lators of cell death. Blood, 80: 879-886, 1992.

1 1 . Li, M., Hu, J. Heermeier, K., Hennighausen, L, and Furth, P. A.Apoptosis and remodeling of mammary gland tissue during involutionproceeds through p53-independent pathways. Cell Growth & Differ., 7:13-20, 1996.

12. Hennighausen, L G., and Sippel, A. E. Characterization and cloning of

the mRNAs specific for the lactating mouse mammary gland. Eur. J.

Biochem., 125: 131-141, 1982.

13. Morrison, B., and Leder, P. Neu and ras initiate murine mammary

tumors that share genetic markers generally absent in c-myc and int-2initiated tumors. Oncogene, 9: 3417-3426, 1994.

14. Daniel, C. W., and Robinson, S. D. Regulation of mammary growth

and function by TGF-�. Mol. Reprod. Dcv., 32: 145-151 , 1992.

15. Binas, B., Spitzer, E., Zschiefche, W., Erdmann, B., Kurtz, S., Muller,P., Niemann, C., Blenau, W., and Grosse, A. Hormonal induction of

functional differentiation and mammary-derived growth inhibitor expres-sion in cultured mouse mammary gland explants. In Vitro Cell. Dev. Biol.,28A: 625-634, 1992.

16. Merlo, G. A., Basolo, F., Fiore, L, Duboc, L, and Hynes, N. E.

p53-dependent and p53-independent activation of apoptosis in mam-mary epithelial cells reveals a survival function of EGF and insulin. J. Cell

Biol., 128: 1185-1196, 1995.

Cell Growth & Differentiation 11

1 7. Strasser, A., Harris, A. W., Jacks, T., and Cory, S. DNA damage caninduce apoptosis in proliferating lymphoid cells via p53-independent

mechanisms inhibitable by bcl-2. Cell, 79: 329-339, 1994.

18. Sedlak, T. W., Zoltan, N. 0., Yang, E., Wang, K., Boise, L. H.,Thompson, C. B., and Korsmeyer, S. J. Multiple BcI-2 family members

demonstrate selective dimerization with Bax. Proc. NatI. Acad. Sci.

USA, 92: 7834-7838, 1995.

19. Oltvai, Z. N., and Korsmeyer, S. J. Checkpoints of dueling dimers foildeath wishes. Cell, 79: 189-192, 1994.

20. Oltvai, Z. N., Milliman, L L, and Korsmeyer, S. Bcl-2 heterodimerizesin vivo with a conserved homolog, bax, that accelerates programmed cell

death. Cell, 74: 609-619, 1993.

21 . Boise, L H., Gonzalez-Garcia, M., Postema, C. E., Ding, L, Lindsten,T., Turka, L A., Mao, x., Nunez, G., and Thompson, C. A. bcl-x, a bcl-2related gene that functions as a dominant regulator of apoptotic celldeath. Cell, 74: 597-608, 1993.

22. Gonzalez-Garcia, M., Perez-Ballestero, R., Ding, L, Buan, L, Boise,

L, Thompson, C., and Nunez, G. Bcl-xL is the major bcl-x mRNA formexpressed during murine development and its product localizes to mito-

chondria. Development (Camb.), 120: 3033-3042, 1994.

23. Miyashita, T., and Reed, J. C. Tumor suppressor p53 is a directtranscriptional activator of the human bax gene. Cell, 80: 293-299, 1995.

24. Schneider, J. W., Gu, W., Zhu, L, Mahdavi, W., and Nadal-Ginard, B.Reversal of terminal differentiation mediated by p107 in Rb-/- muscle

cells. Science (Washington DC), 264: 1467-1471 , 1994.

25. Furth, P. A., St. Onge, L, Bager, H., Gruss, P., Gossen, M., Kistner,A., Bujard, H., and Hennighausen, L Temporal control of gene expressionin transgenic mice by a tetracycline responsive promoter in transgenic

mice. Proc. NatI. Acad. Sci. USA, 91: 9302-9306, 1994.

26. Hennighausen, L, Wall, R. J., Tillmann, U., Li, M., and Furth, P. A.

Conditional gene expression in secretory tissues and skin of transgenicmice using the MMTV-LTR and the tetracycline responsive system. J. Cell.

Biochem., 59: 463-472, 1995.

27. Basola, F., Fiore, L, Ciardiello, F., Calvo, S., Fontanini, G., Conaldi,

P. G., and Toniolo, A. Response of normal and oncogene-transformed

human mammary epithelial cells to transforming growth factor (31 (TGF-�31): lack ofgrowth-inhibitory effect on cells expressing the simian virus 40

large-T antigen. Int. J. Cancer, 56: 736-742, 1994.

28. Franch, H. A., Shay, J. W., Alpem, A. J., and Preisig, P. A. Involvement

of pRb family in TGF�3-dependent epithelial cell hypertrophy. J. Cell Biol.,

129: 245-254, 1995.

29. Schwarz, J. K., Bassing, C. H., Kovesdi, I., Datto, M. B., Blazing, M.,

George, S., Wang, X-F., and Nevins, J. A. Expression of the E2F1 tran-

scription factor overcomes type (3 transforming growth factor-mediatedgrowth suppression. Proc. NatI. Acad. Sci. USA, 92: 483-487, 1995.

30. Adamczewski, J. P., Gannon, J. V., and Hunt, T. Simian virus 40 largeT antigen associates with cyclin A and p33cdk2. J. Virol., 67: 6551-6557,

1993.

31 . Wang, W. B., Bikel, I., Marsilio, E., Newsome, D., and Livingston,

D. M. Transrepression of RNA polymerase II promoters by the simian virus

40 small T antigen. J. Virol., 68: 6180-6187, 1994.

32. Rajan, P., Swaminathan, S., Zhu, J., Cole, C. N., Barber, G., Tevethia,

M. J., and Thimmapaya, B. A novel translational regulation function for the

simian virus large-T antigen gene. J. Virol., 69: 785-795, 1995.

33. Gilinger, G., and Alwine, J. C. Transcriptional activation by simian

virus 40 large T antigen: requirements for simple promoter constructs

containing either TATA or initiator elements with variable upstream factorbinding sites. J. Virol., 67: 6682-6688, 1993.

34. Rice, P. W., and Cole, C. N. Efficient transcriptional activation of manysimple modular promoters by simian virus 40 large T antigen. J. Virol., 67:

6689-6697, 1993.

35. Shamay, A., Pursel, V. G., Wilkinson, E., Wall, R. J., and

Hennighausen, L Expression of the whey acidic protein in transgenic pigs

impairs mammary development. Transgenic Res., 1: 124-132, 1992.

36. Chomczynski, P., and Sacchi, N. Single step method of RNA isolation

by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.

Biochem., 162: 156-159, 1987.