cell cycling predicts p53...

INTRODUCTION

The multiphasic post-natal developmental processes occurringin the murine mammary gland are exquisitely orchestrated byendocrine regulation. Circulating steroid hormones estrogenand progesterone, act on the mammary gland to cause rapidductal elongation and infiltration into the surrounding adiposetissue, ultimately establishing the mature resting gland (Nandiet al., 1958; Delouis et al., 1980; Ceriani et al., 1974;Woodward et al., 1998). Subsequent to maturation, thequiescent gland responds to the hormones of pregnancy,specifically estrogen, progesterone, placental lactogen andprolactin, with an initial burst of epithelial cell proliferation onday 4 of pregnancy, followed by a second proliferative peak atapproximately day 12 (Traurig et al., 1967). By this time,circulating levels of the lactogenic hormone prolactin promotetranscription of β-casein mRNA, the hallmark of mammaryepithelial differentiation, as synthesis of milk proteins begins(Robinson et al., 1995). At parturition, lactation is establishedand reaches maximum yield by day 5. Within 10 days of

weaning, the mammary gland undergoes massive structuralremodeling during which over 80% of its differentiatedepithelium is deleted by apoptosis (Walker et al., 1989; Strangeet al., 1992). These developmental phases in the murinemammary gland mimic closely the stages of human breastdevelopment (Cardiff et al., 1999), making it a useful model ofboth mammary gland development and susceptibility totumorigenesis (Medina, 1996; Ghebranious and Donehower,1998).

Numerous carcinogens may initiate cellular transformation(Otteneder and Lutz, 1999; Vineis et al., 1999), and exposureto ionizing radiation is one of the most widely studied (Ullrich,1991; Barcellos-Hoff, 1998; Ron, 1998; Little, 2000).Epidemiological studies of women exposed to ionizingradiation suggest there may be a period of mammary glanddevelopment, subsequent to the adolescent proliferative phasebut prior to a first pregnancy, during which the mature,quiescent mammary gland is particularly susceptible to γ-irradiation-induced tumorigenesis (Land et al., 1980;Aisenberg et al., 1997).

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The tumor suppressor gene, TP53, plays a major role insurveillance and repair of radiation-induced DNA damage.In multiple cell types, including mammary epithelial cells,abrogation of p53 (encoded by Trp53) function is associatedwith increased tumorigenesis. We examined γ-irradiatedBALB/c-Trp53+/+ and -Trp53–/– female mice at five stages ofpost-natal mammary gland development to determinewhether radiation-induced p53 activity is developmentallyregulated. Our results show that p53-mediated responsesare attenuated in glands from irradiated virgin andlactating mice, as measured by induction of p21/WAF1(encoded by Cdkn1a) and apoptosis, while irradiated early-and mid-pregnancy glands exhibit robust p53 activity.There is a strong correlation between p53-mediatedapoptosis and the degree of cellular proliferation,independent of the level of differentiation. In vivo,proliferation is intimately influenced by steroid hormones.To determine whether steroid hormones directly modulate

p53 activity, whole organ cultures of mammary glands wereinduced to proliferate using estrogen plus progesterone orepidermal growth factor plus transforming growth factor-α and p53 responses to γ-irradiation were measured.Regardless of mitogens used, proliferating mammaryepithelial cells show comparable p53 responses to γ-irradiation, including expression of nuclear p53 andp21/WAF1 and increased levels of apoptosis, compared tonon-proliferating irradiated control cultures. Our studysuggests that differences in radiation-induced p53 activityduring post-natal mammary gland development areinfluenced by the proliferative state of the gland, and maybe mediated indirectly by the mitogenic actions of steroidhormones in vivo.

Key words: p53, γ-irradiation, Mammary gland, Apoptosis, Mouse,Human

SUMMARY

DEVELOPMENT AND DISEASE

Epithelial cell cycling predicts p53 responsiveness to γ-irradiation during

post-natal mammary gland development

Lisa M. Minter 1, Ellen S. Dickinson 1, Stephen P. Naber 2 and D. Joseph Jerry 1,*1Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA2Department of Pathology, Baystate Medical Center, Springfield, MA, USA *Author for correspondence (e-mail: [email protected])

Accepted 19 March 2002

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Mammalian cells possess surveillance mechanisms to detectand repair radiation-induced DNA damage, one of the mostimportant being the tumor suppresser gene, TP53 (Levine etal., 1991; Cox and Lane, 1995; Ko and Prives, 1996; Levine,1997). Functional p53 is not essential for survival to adulthood,but mice homozygous for the Trp53–/– allele show increasedincidence of a spectrum of tumors (Donehower et al., 1992).As many as 40% of human breast cancers (Coles et al., 1992)and up to 50% of all cancers exhibit mutations in TP53, makingit the most commonly mutated gene in human cancers (Levineet al., 1991). Activated p53 can mediate cell-cycle arrestas well repair of radiation-induced double-strand breaks inDNA (reviewed by Amundson et al., 1998). Post-translationalmodifications of p53 facilitate its stabilization, nuclearaccumulation, and increased half-life (Siliciano et al., 1997),promoting transcription of downstream target genes includingp21WAF1 and Mdm2.

Biological consequences of p53 activity include cell cyclearrest, induction of senescence, or apoptosis, depending uponwhat additional signals are received and within what context.Studies of both embryonic and adult murine tissues havedemonstrated tight regulation of Trp53activation following γ-irradiation, and suggest that these responses are not only celltype-specific, but may also be developmentally regulated(Merritt et al., 1994; Midgley et al., 1995; MacCallum et al.,1996).

Despite the critical role p53 plays in suppressing radiation-initiated transformation, and studies suggesting the mature,quiescent breast is particularly sensitive to radiation-inducedtumorigenesis, little is known about p53 responses to ionizingradiation in the adult mammary gland across its post-nataldevelopmental spectrum. Here we show that p53-mediatedresponses to γ-irradiation in mammary epithelial cells (MECs)vary during post-adolescent mammary gland development, andthat these responses are influenced by the degree of proliferationintrinsic to specific developmental stages, not by theaccompanying level of differentiation. Furthermore, wedemonstrate that MECs stimulated to proliferate in the presenceor absence of steroid hormones show equivalent p53-mediatedresponses to ionizing radiation. Together these data suggest thatradiation-induced p53 activation is greatest during peakproliferative stages of mammary gland development and may bemediated in vivo by the mitogenic actions of steroid hormones.

MATERIALS AND METHODS

Animal and tissue proceduresFourth inguinal mammary glands were isolated from age-matched 8-to 12-week old virgin (V), day-4 pregnant (P4), day-15 pregnant(P15), day-5 lactating (L5) and day-7 weaning (W7) BALB/c-Trp53+/+ and -Trp53–/– female mice before, or 6 hours after treatmentwith 5 Gy of whole body irradiation (n=3 per developmentalstage/genotype/radiation group). Day of gestation was determined bycounting forward from the appearance of a vaginal plug, which wascounted as day one. Radiation was administered using a 137Csirradiator. Six hours after γ-irradiation, tissues were fixed overnight in10% phosphate-buffered formalin (PBF), rinsed in 70% ethanol, andparaffin-embedded.

ImmunohistochemistryFor detection of p53 and p21/WAF1, 4 µm sections of mammary

tissue were deparaffinized in xylene, rehydrated in graded ethanol,rinsed in phosphate-buffered saline (PBS) then subjected tomicrowave antigen retrieval in 0.01 M citrate buffer. Tissues wereincubated in 0.3% hydrogen peroxide (Sigma Chemicals, St. Louis,MO) in methanol, blocked with 5% nucleic acid blocker (RocheDiagnostics Corp., Indianapolis, IN) in 0.1 M maleate buffer, andincubated overnight at 4°C with rabbit polyclonal CM5 anti-p53antiserum (1:1500, Novacastra, Newcastle upon Tyne, UK) orrabbit polyclonal Ab-5 anti-p21/WAF1 antiserum (1:50, OncogeneResearch Products, Cambridge, MA). Negative controls for p53immunohistochemistry consisted of mammary tissue from age- andtreatment-matched Trp53–/– female mice. Omitting primary antibodyfrom staining procedures generated negative controls for p21/WAF1and proliferating cell nuclear antigen (PCNA) immunohistochemistry.The mouse monoclonal PC10 anti-PCNA antibody (1:50, BDPharmingen, San Diego, CA) and the MOM Detection Kit (VectorLaboratories, Burlingame, CA) were used according to themanufacturer’s directions to detect PCNA expression in 4 µm sectionsof mammary tissue that had been subjected to microwave antigenretrieval. Immunolabeled complexes were amplified using the VectorABC Elite avidin-biotin-horseradish peroxidase labeling kit (VectorLaboratories) and 3,3′-diaminobenzidine (Sigma). Tissue sectionswere counterstained with Methyl Green, dehydrated through gradedethanol and xylene, and cover-slipped. The percentage of cells cyclingwas determined using the BioQuant Imaging Software System (R&MBiometrics, Inc., Nashville, TN). For in vivo glands, a minimum of1500 mammary epithelial cells was counted (3 animals pertreatment/developmental stage, 6-8 fields/animal). For whole organcultures, a minimum of 1200 mammary epithelial cells was countedper treatment (600 cells per culture/duplicate cultures per treatment).Differences in the percentage of cells cycling between treatments weredetermined using a test of 2 proportions with a 95% confidenceinterval.

In situ end-labeling of DNAFour µm sections of mammary tissue were deparaffinized, incubatedin 0.3% hydrogen peroxide in methanol, and subjected to terminaldeoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) ofapoptotic DNA, using the FragEL DNA Fragmentation Kit (OncogeneResearch Products). Sections were counterstained with Methyl Greenand the percentage of apoptotic cells was determined using theBioQuant Imaging Software System. For in vivo glands, a minimumof 2400 mammary epithelial cells and 10,000 lymphocytes fromintramammary lymph nodes were counted per treatment/perdevelopmental stage (3 animals per treatment/developmental stage, 2-4 fields/animal). For whole organ cultures, a minimum of 1200mammary epithelial cells was counted per treatment (600 cells perculture/duplicate cultures per treatment). Differences in thepercentage of TUNEL-positive cells among treatments werecompared using a test of 2 proportions with a 95% confidence interval.

Northern blot hybridizationFourth inguinal mammary glands from unirradiated V, P4, P15, L5and W7 BALB/c-Trp53+/+ female mice were homogenized inUltraspec RNA (BioTecx Laboratories, Houston, TX), 1 ml/100 mgof tissue, before RNA isolation using a modified Chomczynskimethod (Chomczynski and Sacchi, 1987). Ten µg of total RNA wasseparated on a 1.2% agarose-formaldehyde gel, transferred to a nylonmembrane (Zetabind; CUNO, Meriden, CT) and hybridized with a32P-labeled β-casein cDNA probe. The blot was then stripped andreprobed with a 32P-labeled GAPDH cDNA probe to demonstrateequivalent sample loading.

Whole organ culturesFourth inguinal mammary glands from age-matched, unprimed, 8- to12-week old virgin BALB/c-Trp53+/+ and -Trp53–/– female mice wereremoved aseptically, cut in half then floated on siliconized lens paper

L. M. Minter and others

2999Cell cycling predicts p53 responsiveness

in 35 mm tissue culture dishes containing 2 ml of medium. Controlmedium consisted of serum-free DMEM/F12 buffered with Hepes andNaHCO3, plus insulin (5 µg/ml; GibcoBRL Life Technologies, GrandIsland, NY). Hormone-supplemented medium was prepared byadding 1 ng/ml 17-β-estradiol (Sigma) and 1 µg/ml progesterone(Sigma) to control medium. Growth factor (GF)-supplementedmedium was prepared by adding 20 ng/ml each of EGF (Sigma) andTGFα (R&D Systems, Minneapolis, MN) to control medium. Wholeorgan cultures (WOCs) were maintained for 96 hours in 5% CO2 inair, with daily media changes. After 96 hours, WOCs were subjectedto 5 Gy of γ-radiation, using a 137Cs irradiator, while control WOCswere sham irradiated. Six hours after γ-irradiation, all WOCs werefixed in 10% PBF overnight, rinsed in 70% ethanol and paraffin-embedded.

RESULTS

Nuclear p53 is differentially expressed in the post-natal mammary gland following γ-irradiationWe recently demonstrated that virgin mammary glands ofTrp53+/+ mice fail to mount p53-mediated responses to γ-irradiation, but upon transient treatment with pregnancy-associated hormones, a robust radiation-induced p53 responseis observed (Kuperwasser et al., 2000). This suggested thatradiation-induced p53 responses might be developmentallyregulated in the post-natal mammary gland. To examine this

possibility, we treated age-matched virgin (V), mid-pregnant(P15), lactating (L5) and weaning (W7) Trp53+/+ and Trp53–/–

female mice with 5 Gy of whole body radiation and comparedtheir p53 responses to that of unirradiated mice usingimmunohistochemical approaches. Pilot studies of radiationresponses at 10 and 30 minutes, and 1, 6 and 24 hours post-irradiation determined the 6-hour timepoint to be optimal formulti-parameter measurements. While nuclear p53 wasdetectable 1 hour following irradiation, p21/WAF1 expressionpeaked at 6 hours, consistent with the kinetics of the p53radiation response (MacCallum et al., 1996) (C. Kuperwasser,unpublished results). Additionally, TUNEL staining performedat early timepoints detects double-strand DNA breaks stillundergoing repair that, by 6 hours post-irradiation, are thehallmark of apoptotic cells. Furthermore, analysis at 24 hourspost-irradiation did not show significant increases in cell deathin irradiated virgin glands (data not shown), ruling out adelayed induction of apoptosis as a mechanism for thedifferential apoptotic responses observed across thedevelopmental spectrum.

Nuclear accumulation of p53 is requisite for its biochemicaland biological activities (Martinez et al., 1997; Middeler et al.,1997). In unirradiated control Trp53+/+ female mice, nuclearp53 was not detected by immunohistochemical staining in theepithelium of mammary glands at the four stages of post-natalmammary gland development analyzed (Fig. 1A-D). In

Fig. 1.Radiation-induced p53 protein expression varies during mammary gland development. Immunohistochemistry using CM5 anti-p53antisera was performed on BALB/c-Trp53+/+ mammary tissue before (unirradiated, A-D) or 6 hours after (irradiated, E-H) treatment with 5 Gyof whole-body γ-radiation at various stages of post-natal development. Irradiated age-matched, and developmental stage-matched BALB/c-Trp53–/– mice served as controls (I-L). (Magnification 40×).

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contrast, following γ-irradiation of Trp53+/+ female mice,robust nuclear p53 staining was observed in many ductalepithelial cells of P15 glands (Fig. 1F), while in W7 tissuesstrong nuclear staining was seen in a moderate number ofductal epithelial cells (Fig. 1H). However, irradiated V and L5glands showed little or no nuclear p53 staining in ductalepithelium (Fig. 1E,G), stromal adipocytes, or periductalfibroblasts. Nuclear p53 could be seen in irradiatedlymphocytes of the intramammary lymph node at all stages ofdevelopment (data not shown) and was used as an internalcontrol verifying radiation delivery.

Non-specific staining or cross-reactivity of the CM5antibody with nuclear proteins can be excluded since irradiatedmammary epithelium from age- and developmental stage-matched Trp53–/– mice were devoid of nuclear p53 staining(Fig. 1I-L). These data show that expression of radiation-induced nuclear p53 varies in a cell type-specific mannerwithin the mammary gland, and within the epithelialcompartment of the gland, nuclear p53 expression varies acrossthe developmental spectrum.

Nuclear localization of p53 correlates withtransactivation of its target gene, p21WAF1

Transcriptional activity of p53 following γ-irradiation can bemeasured by induction of downstream target genes, including

p21WAF1, Bax and GADD45(el-Deiry, 1998; Bouvard, 2000).We sought to determine the biochemical activity of nuclear p53by assessing its ability to transactivate p21WAF1, as its inductionin the mammary gland was shown to be p53 dependent (Jerryet al., 1998). Basal levels of p21/WAF1 protein expressionwere uniformly low in mammary tissues of unirradiated controlTrp53+/+ (Fig. 2A-D) and Trp53–/– female mice (data notshown), regardless of developmental stage. However,following irradiation, strong nuclear staining of p21/WAF1was observed in many ductal epithelial cells of Trp53+/+ P15mammary glands and also at lower frequency in W7 glands(Fig. 2F,H). Lymphocytes of intramammary lymph nodes alsoshowed robust nuclear p21/WAF1 staining (data not shown).Mammary epithelium from Trp53+/+ V and L5 glands exhibitedlittle to no nuclear p21/WAF1 staining (Fig. 2E,G), thoughnuclear p21/WAF1 staining was evident in the irradiatedlymphocytes of their intramammary lymph nodes at levelscomparable to P15 and W7 mammary tissues (data not shown).Consistent with their lack of nuclear p53 expression, stromaladipocytes and periductal fibroblasts failed to show significantnuclear p21/WAF1 staining at any stage of development incontrol or irradiated wild-type tissues.

Mammary tissues from irradiated Trp53–/– mice were devoidof nuclear p21/WAF1 expression at all stages of development(Fig. 2I-L), demonstrating the p53-dependent nature of the


Fig. 2.Radiation-induced p21/WAF1 protein expression varies during mammary gland development. Immunohistochemistry using Ab5 anti-p21/WAF1 antisera was performed on BALB/c-Trp53+/+ mammary tissue before (unirradiated, A-D) or 6 hours after (irradiated, E-H) treatmentwith 5 Gy of whole-body γ-radiation at various stages of post-natal development. Irradiated age-matched, and developmental stage-matchedBALB/c-Trp53–/– mice served as controls (I-L). (Magnification 40×).


response in Trp53+/+ mice. Thus, γ-radiation-inducedtranscriptional activity of p53 varies during post-natal mammarygland development, and parallels its nuclear localization.

Activated p53 mediates biological responses atdistinct stages of developmentCell cycle arrest and apoptosis are biological responses to γ-irradiation mediated by p53 in a variety of cell types (Kastanet al., 1991; Lowe et al., 1993a; MacCallum et al., 1996; Meyeret al., 1999; Komarova et al., 2000; D’Sa-Eipper et al., 2001).Basal apoptotic indices in mammary epithelium fromunirradiated control Trp53+/+ and Trp53–/– female mice wereless than 1% at each of the four stages of post-natal mammarygland development examined, consistent with previouslypublished results (Meyn et al., 1996; Kuperwasser et al., 2000),while levels of apoptosis in the mammary epithelium ofirradiated Trp53+/+ mice varied with stage of development andclosely paralleled the expression of nuclear p53 (Fig. 3). In theirradiated V gland, there was a small but significant increasein epithelial cell apoptosis above basal levels (3.4% vs. 0.92%,P<0.05) while no increase was seen in L5 mammary tissue(0.82% vs. 0.37%, P>0.05). In contrast, a robust and significantincrease in apoptosis was seen in the irradiated epithelium ofthe P15 gland (11.73% vs. 0.55%, P<0.05). The irradiated W7gland displayed an intermediate level of apoptosis (4.69% vs.0.57%, P<0.05), significantly higher than basal levels but lowerthan mid-pregnant values. Unlike the mammary epithelial cellcompartment, the percentage of apoptotic lymphocytes in theintramammary lymph nodes of irradiated Trp53+/+ mammary

glands remained consistently high regardless of developmentalstage (mean=20.7%±5.64%).

The mammary epithelium of irradiated Trp53–/– female miceshowed a distinct absence of cell death, as did lymphocytes ofintramammary lymph nodes at all stages of developmentexamined (data not shown), confirming the essential role ofp53 in radiation-induced apoptosis in these tissues. Therefore,as with its biochemical activity, the biological activity of p53in the mammary gland varies with the developmental stage ofthe gland and is cell-type specific.

Proliferation, not differentiation, influencesradiation-induced p53 activityMammary glands from virgin and mid-pregnant mice representdiverse stages of post-natal development, both morphologicallyand functionally. Specifically, the mid-pregnancy (P15) glandexhibits a significantly increased proliferative capacity as itresponds to the hormonal milieu of pregnancy. Furthermore,by day 15 of pregnancy, the mammary epithelium has initiatedthe genetic program that drives it toward its fully differentiated,lactating state (Robinson et al., 1995). We sought to distinguishwhich of these, proliferation or differentiation, might influencep53 response to γ-irradiation, and therefore chose to examineradiation-induced p53 responses in mammary glands of 4-daypregnant (P4) mice, which show increased proliferation, but anabsence of differentiation.

To quantify the degree of stage-specific proliferation at thetime of irradiation, we immunolabeled proliferating cellnuclear antigen (PCNA) in control and irradiated mammarytissues of virgin, P4 and P15 mice. In non-quiescent cells,PCNA can be detected during all phases of the cell cycle, butits expression increases during S-phase (Celis and Celis, 1985).In contrast, BrdU will only be incorporated into cells thatsuccessfully transit the S-phase. Thus, to use BrdU labeling asa means of identifying cycling cells would potentiallyeliminate from quantitation those cells arresting at the G1/Scheckpoint, as well as cells in G2 during the radiation eventand subsequently arresting at G2/M, both points at which p53has been shown to mediate cell cycle-arrest (Kastan et al.,1991; Yonish-Rouach et al., 1993; Morgan and Kastan, 1997).Quantitation of PCNA, therefore, reflects a more inclusiveprofile of cycling mammary epithelial cells. The percentage ofPCNA-positive cells in the V mammary gland was significantlylower than either the P4 or P15 gland (5.86% vs 18.4% and9.46%, respectively, P<0.05; Fig. 4A,Bi-iii). Additionally,there is strong correlation between the percentage of cyclingcells and the percentage of apoptotic cells in the irradiatedepithelium of V, P4 and P15 glands, but not in unirradiatedcontrol glands (Fig. 4A,Biv-vi; data not shown).

Our data, therefore, confirm the proliferative burst intrinsicto the P4 mammary gland and induced by the hormonal stimuliof early pregnancy. Following γ-irradiation, and similar to theP15 gland, the epithelium of the P4 mammary gland showsstrong induction of nuclear p53, as well as nuclear p21/WAF1(Fig. 5Aiii,iv). Yet unlike the P15 developmental stage, the P4gland maintains its undifferentiated state as measured byinduction of β-casein mRNA (Fig. 5B), the first lactation-essential milk protein to be upregulated during pregnancy(Robinson et al., 1995). Together, these data suggest that p53-mediated responses to γ-irradiation are greatest in theproliferating epithelium of the mammary gland, and the

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Fig. 3.Radiation-induced apoptosis of mammary epithelium variesduring post-natal development. The percentage of apoptotic cells inmammary epithelium and intramammary lymph nodes was measuredby TUNEL assay on BALB/c-Trp53+/+ mammary tissues before(control) or 6 hours after (irradiated) treatment with 5 Gy of whole-body γ-radiation at various stages of post-natal development.Asterisks indicate values significantly different from unirradiatedcontrols (P<0.05). Error bars represent s.e.m.

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robustness of the responses seen is dictated by the extent ofproliferation, not differentiation.

Proliferative stimuli render p53 competent torespond to γ-irradiationIn vivo, proliferation in the mammary gland is intimatelyinfluenced by steroid hormones. Studies suggest this action isindirect or paracrine in nature, and may be mediated by themitogenic effects of growth factors released by epithelial andstromal cells in response to steroid hormones (Woodward etal., 1998). As such, we sought to distinguish direct hormonalmodulation of p53 activity from any indirect effects on p53activation, as mediated by the proliferative actions of steroidhormones. To do so, we induced the epithelial cellcompartment of whole organ cultures (WOCs) to proliferate inthe presence or absence of the combined steroid hormones,

estrogen plus progesterone, then irradiated the glands andmeasured p53-dependent responses. WOCs cultured in thepresence of estrogen (1 ng/ml) plus progesterone (1 µg/ml) butwithout growth factors, or in the presence of EGF (20 ng/ml)plus TGFα (20 ng/ml) but without steroid hormones, showedsmall but significant increases in the percentage of cells cycling(3.57% and 3.45%, respectively; P<0.05) compared to controlglands grown in the absence of both hormones and growthfactors (0.98%). These increases were also consistent withprevious studies (Spitzer et al., 1995). After treatment with 5Gy of γ-radiation, nuclear localization of p53 and p21/WAF1could be detected in luminal epithelial cells of WOCsmaintained in the presence of either hormones or growthfactors (Fig. 6B,C and E,F, respectively), but not in thosegrown in control medium (Fig. 6A,D). Additionally, thepercentage of apoptotic cells seen in irradiated WOCs cultured


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Fig. 4. Apoptosis correlates with PCNA expressionin irradiated mammary glands. (A) The percentage ofcycling cells was determined immunohistochemicallyby calculating the percentage of PCNA-positiveepithelial cells, while the percentage of apoptoticcells was determined by TUNEL assay on virgin (V),early- (P4) and mid-pregnancy (P15) BALB/c-Trp53+/+ mammary tissues before (control) or 6hours after (irradiated) treatment with 5 Gy of whole-body γ-radiation. Asterisks indicate valuesstatistically different from unirradiated controls(P<0.05). Error bars represent s.e.m.(B) Immunohistochemistry was performed usingPC10 anti-PCNA antibody (i-iii) while apoptosis wasmeasured by TUNEL staining (iv-vi) on virgin (V),early- (P4) and mid-pregnancy (P15) BALB/c-Trp53+/+ mammary tissues 6 hours after treatmentwith 5 Gy of whole-body γ-radiation. (Magnification40×).


in either hormone- or growth factor-supplemented medium wassignificantly higher than irradiated control WOCs, andcorrelated closely with the percentage of cycling cells underthese growth conditions (Fig. 7). Thus, induction of a p53-dependent response to γ-irradiation in MECs from unprimedvirgin mice could be achieved using growth factors to stimulatetheir proliferation prior to irradiation. These data suggest thatdirect hormonal modulation of p53 is not required to activatep53 in response to ionizing radiation.

DISCUSSION

Radiation-induced p53 activity varies during post-natal development Our initial set of experiments revealed an interesting andsomewhat unexpected profile of p53-mediated responses to γ-irradiation. Nuclear localization of p53 protein correlated withits biochemical activity, and this correlation could bedemonstrated in both epithelial and lymphocytic cells, whichdisplayed parallel expression/activity profiles. Likewise,significant induction of apoptosis was only seen in stages ofmammary gland development that also exhibited accumulationof nuclear p53. These observations are in agreement withprevious work showing transcriptional activity of p53 isrequired to mediate its apoptotic responses (Jiminez etal., 2000; Zhu et al., 1998). Additionally, our evidencethat nuclear p53 accumulates in some cell types withinthe heterogeneous mammary gland but not others, isalso consistent with earlier studies demonstrating celltype-specific induction of p53 in response to γ-irradiation (Midgley et al., 1995).

Surprisingly, the p53 responses to γ-irradiationexhibited by mammary epithelial cells differeddramatically across the post-natal developmentalspectrum. Despite the fact that these cells areintrinsically similar, there appear to be sufficientdifferences in ontogeny to significantly alter nuclearaccumulation of p53 and its concomitant functionalactivity following γ-irradiation. While unexpected, thisis not entirely without precedence, as MacCallum etal. (MacCallum et al., 1996) also demonstrateddevelopmentally regulated differences in tissue-specificp53-mediated radiation responses.

The data presented here confirm and extend the workof Kuperwasser et al. (Kuperwasser et al., 2000)demonstrating attenuated p53 responses to γ-irradiationin the mammary epithelium of Trp53+/+ virgin mice. Inaddition, the present study demonstrates that stronginduction of p53 in the mammary epithelium ofirradiated mid-pregnant Trp53+/+ mice is associatedwith significant increases in apoptosis. In someradiation-sensitive tissues such as splenic white pulpand the bases of the crypts in the small intestine, thepresence of high levels of Trp53 mRNA have beensuggested as a factor influencing radiation-inducedapoptotic responses (Komarova et al., 2000). Unlikethese tissues, however, the differential accumulation ofnuclear p53 detected in irradiated P15 glands cannot beattributed to differences in Trp53 mRNA levelsbetween virgin and mid-pregnant stages, since the

presence of similarly high levels of Trp53 transcript in glandsof virgin, mid-pregnant and weaning females has beenpreviously demonstrated (Pinkas and Jerry, unpublishedresults) (Jerry et al., 1998; Kuperwasser et al., 2000). It isdoubtful, therefore, that the observed p53-mediated responsesto radiation in the mammary gland are modulated at thetranscriptional level.

As a consequence of cell death, apoptotic cells are identifiedand efficiently removed by phagocytes (reviewed by Fadok andChimini, 2001). Because the underlying principle of TUNELlabeling precludes its use as an early indicator of apoptosis,accelerated clearance of apoptotic cells in V and L5 glandscannot be entirely ruled out. However, studies on the kineticsof apoptotic cell clearance show that while the minimumamount of time needed for clearance may be several hours,DNA fragmentation, such as detected by TUNEL staining, canprecede clearance by up to several days (Blaschke et al., 1996;Pompeiano et al., 1998), suggesting that rapid clearance is anunlikely explanation for the disparate levels of apoptosisobserved during mammary gland development.

At the protein level, mutation and nuclear exclusion haveboth been shown to result in compromised p53 activity.Cytoplasmic sequestration of p53 in mammary epithelial cellshas been postulated as one mechanism that may promote breast

Fig. 5. p53 activity but not β-caseinis abundant in irradiated early-pregnancy (P4) mammary glands.(A) Immunohistochemistry usingCM5 anti-p53 (i,iii) and Ab5 anti-p21/WAF1 (ii,iv) antisera wasperformed on early-pregnancy (P4)BALB/c-Trp53+/+ mammary tissuebefore (unirradiated) or 6 hoursafter (irradiated) treatment with 5

Gy of whole-body γ-radiation. (Magnification 40×). (B) Northern blot analysisof duplicate total RNA samples (10 µg) from fourth inguinal mammary glandsof BALB/c-Trp53+/+ mice were hybridized with a cDNA probe of β-casein(upper panel). Lower panel shows GAPDH expression as loading control.

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tumorigenesis (Moll et al., 1992; Kuperwasser et al., 2000),however, unlike the findings of Kuperwasser et al., cytoplasmicp53 in mammary epithelium from control or irradiatedTrp53+/+ virgin mice was not detected in this study. Thereasons for this inconsistency are unclear, but it may reflectintrinsic lot-to-lot variability of epitopes in polyclonal antiserafrom different animals, and the significantly lower antibodyconcentrations used in our studies (1:1500 vs. 1:200).However, in agreement with Kuperwasser’s results, no nuclearp53 or p21/WAF1 was detected, nor was significant inductionof apoptosis observed in the mammary epithelial cells ofirradiated virgin Trp53+/+ mice.

This study not only confirms that the mammary gland ofvirgin mice represents a stage of post-natal developmentcharacterized by attenuated p53 responses to ionizingradiation, but also reveals that the lactating gland is similarlyrefractory. In contrast, glands of pregnant and weaning miceshow significant p53-mediated responses to ionizingradiation.

Apoptotic response to γ-irradiation correlates withextent of epithelial cell proliferationDuring early embryogenesis, many rapidly proliferating tissueshave been shown to accumulate high levels of p53 proteinfollowing γ-irradiation, without undergoing apoptosis,suggesting p53 may be functioning to prevent teratogenesisand preserve developmental plasticity (MacCallum et al.,1996). As gestation proceeds, clear heterogeneity of p53induction and of p53-mediated apoptotic responses to γ-irradiation become apparent, suggesting that p53 may assumea quite different role in fully developed tissues. In the adult


Fig. 6. p53 and p21/WAF1 expression are induced in proliferating, γ-irradiated whole organ cultures. Immunohistochemistry using CM5 anti-p53 (A-C) and Ab5 anti-p21/WAF1 (D-F) antisera was performed on whole organ cultures maintained for 96 hours in unsupplemented(untreated) medium (A,D), hormone-supplemented (B,E) or growth factor-supplemented (C,F) medium and 6 hours after treatment with 5 Gyof γ-radiation. Hormone-supplemented medium contained estrogen (1 ng/ml) plus progesterone (1 µg/ml), while growth factor-supplementedmedium contained EGF (20 ng/ml) plus TGFα (20 ng/ml). (Magnification 40×.)

0

5

10

Untreated Hormone- Growth Factor- Supplemented Supplemented

Culture conditions

Perc

ent p

ositi

ve c

ells * *

* *

- ++ ++

PCNA

TUNEL

Nuclear p53 localization in irradiated MECs

Fig. 7. Apoptosis correlates with PCNA expression in γ-irradiatedwhole organ cultures. Mammary glands from unprimed virginBALB/c-Trp53+/+ mice were cultured for 96 hours inunsupplemented (untreated), hormone- or growth factor-supplemented medium. Hormone-supplemented medium containedestrogen (1 ng/ml) plus progesterone (1 µg/ml), while growth factor-supplemented medium contained EGF (20 ng/ml) plus TGFα (20ng/ml). The percentage of cycling cells was determinedimmunohistochemically by calculating the percentage of PCNA-positive epithelial cells, while the percentage of apoptotic cells wasdetermined by TUNEL assay on whole organ cultures 6 hours aftertreatment with 5 Gy of γ-radiation. Asterisks indicate valuesstatistically different from irradiated controls in unsupplementedmedium (P<0.05). Error bars represent s.e.m.


animal, in the absence of tissue injury or inflammation,homeostatic cellular replacement in tissues remain relativelyconstant over time (Kumar et al., 2000; Gressner, 2001;Renehan et al., 2001). In many adult tissues, no clearcorrelation between proliferation and p53-mediated responseto irradiation has been demonstrated (MacCallum et al., 1996;Merritt et al., 1994; Midgley et al., 1995). The maturemammary gland is unique, in that it is characterized by definedperiods of extensive cellular proliferation and differentiationduring pregnancy in response to endocrine stimulation, and thisprocess is recapitulated to a much lesser extent during eachestrus cycle (Robinson et al., 1995). In this study, the greatestradiation-induced p53-mediated responses are seen in theearly- and mid-pregnancy mammary glands, where thepercentage of apoptosis in mammary epithelial cells closelycorrelates with the percentage of cells cycling. Relative to thequiescent V gland, both the P4 and P15 mammary glands showextensive epithelial cell proliferation (Fig. 4) with the P4 glandnot yet displaying the hallmarks of differentiation (Fig. 5B).These results suggest it is the degree of proliferation within theepithelial cell compartment that influences p53-mediatedradiation-induced apoptosis in the mammary gland, not thelevel of epithelial cell differentiation. Using an alternative invitro approach, stimulating epithelial cell proliferation inwhole organ cultures with either steroid hormones or growthfactors prior to irradiation, we confirmed the correlationbetween increased cell cycling and increased p53-mediatedapoptotic responses to γ-irradiation. Additionally, thisapproach revealed that the action of steroid hormones on p53-mediated responses to γ-irradiation in mammary epithelial cellsis likely to be indirect, via their mitogenic actions on theepithelium. Taken together, these data suggest that mechanismsintimately associated with proliferation are acting to relieve thefunctional repression of p53 observed in the quiescent Vmammary gland, thus facilitating its activation during periodsof increased mammary epithelial cell cycling.

Regulation of p53 activity is exquisitely complex, and thereare numerous diverse signals capable of activating it. As such,the correlation between mammary epithelial cell proliferationand radiation-induced p53-mediated apoptosis may beexplained by several mechanisms. These include changes insubcellular localization of p53 during the cell cycle, putativephosphorylation of p53 by radiation-responsive kinases thatincrease its stability and half-life, and potential conflicts ingrowth/arrest signals arising from the presence of increasedlevels of proteins required to both drive and halt the cellcycle.

A number of studies have demonstrated cell cycle-dependent, subcellular redistribution of p53, showing that theprotein accumulates in the nucleus during late G1 of the cellcycle and is thus poised to act at the G1/S restriction point(Martinez et al., 1991; Shaulsky et al., 1990; Shaulsky et al.,1991). Importantly, additional studies concluded thatphosphorylation of amino-terminal serine residues, resultingin increased transcription of p53-dependent genes, Mdm2andp21WAF1, was compartmentally restricted and requirednuclear localization of p53 for phosphorylation to occur(Martinez et al., 1997). Redistribution to the nucleus ofcycling cells, places p53 in the proper cellular compartmentto be modified by nuclear kinases such as ATM, which itselfis activated in response to DNA damage induced by ionizing

radiation (Watters et al., 1997). Post-translationalmodification of p53 by ATM and/or DNA-PK results inphosphorylation of specific serine residues in the aminoterminus of the protein, promoting its dissociation fromMdm2, and thus increasing its half-life (Shieh et al., 1997;Mayo et al., 1997). Stabilized p53 may then accumulate inthe nucleus, activating transcription of downstream targetgenes including p21WAF1, GADD45 and Bax, whichorchestrate cell-cycle arrest, facilitate DNA repair, and induceapoptosis, respectively. However, p53 has recently beenplaced in the growth-factor-responsive AKT signalingpathway, which is thought to enhance cellular survival andproliferation by promoting Mdm2-mediated ubiquitinationand degradation of p53 (Mayo et al., 2001; Zhou et al., 2001).While this mechanism may aid proliferation, it is notnecessarily inconsistent with radiation-induced activation ofp53 during stages of rapid cell growth, since cellularresponses to radiation result in modification of both Mdm2and p53 to promote their dissociation and override thenegative regulatory effects of Mdm2 (Mayo et al., 1997).Additionally, the tumor suppressor protein, PTEN, recentlyshown to negatively regulate the AKT pathway, is alsoupregulated by p53 and, following p53 activation, may act tofurther protect it from Mdm2-mediated degradation (Mayo etal., 2002).

Previous studies provide further evidence suggesting a linkbetween cell cycling and cell death. Apoptosis can be inducedin both mouse and rat embryonic fibroblast cell lines co-expressing E2F1 and p53, suggesting that apoptosis can resultfrom a conflict in growth and arrest signals (Wu and Levine,1994). Likewise, in transgenic mice over-expressing E2F1,increased apoptosis and decreased epidermal thickness andcellularity were observed on a Trp53+/– background comparedto their Trp53–/– counterparts, again suggesting conflictingsignals of growth and arrest may be resolved by induction ofapoptosis (Pierce et al., 1998). Consistent with the pattern ofapoptosis we observed in irradiated cycling mammaryepithelial cells of P4 and P15 Trp53+/+ mice, these data arguestrongly in favor of a mechanism of p53-mediated cell deatharising from conflicting signals that concomitantly attempt todrive and halt the cell cycle. Studies are currently underway inour laboratory to elucidate the molecular mechanismsresponsible for regulating radiation-induced p53-mediatedapoptosis in cycling epithelial cells of early- and mid-pregnancy mammary glands that appear to be refractory in thequiescent gland of virgins.

While the pattern of apoptotic responses seen in the γ-irradiated murine mammary gland supports a model ofconflicting growth and arrest signals, other potentialmechanisms may be mediating the observed responses. Bcl2mRNA levels have been shown to be high in the virginmammary gland, relative to levels in the mid-pregnancy gland,while Bax mRNA levels remain fairly constant during post-natal development (J. J., unpublished results). Bcl2 levelscould, therefore, provide overriding survival signals in virginmammary epithelium that are lost as the change in Bcl2/Baxratio favors Bax, and predisposes the mammary epithelium toundergo radiation-induced apoptosis as the gland progressesthrough pregnancy. In the V gland, inactive p53, incapable oftransactivating downstream targets such as Bax, may act tomaintain Bcl2/Baxratios in favor of Bcl2 and survival signals.

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Attenuated p53 function in quiescent mammary epithelialcells may increase susceptibility to radiation-inducedtumorigenesisEpidemiological studies confirm that women exposed toionizing radiation as a result of the Hiroshima bombing,therapeutic fluoroscopies or chest x-rays (Land et al., 1980), orwho received mantle-field irradiation for the treatment ofHodgkin’s disease (Aisenberg et al., 1997), show an increasedrisk of breast cancer relative to the general population. Aunifying corollary of these studies is the inverse risk of cancerwith age at time of exposure to radiation. The present studyhas demonstrated that in the mouse mammary gland, p53responses to ionizing radiation vary during post-adolescentdevelopment, with the quiescent virgin gland exhibiting anattenuated apoptotic response. Decreased p53 response toradiation at this stage of development coincides with the‘window of susceptibility’ that may exist in humans, andduring which the mammary epithelium may be at increasedrisk of radiation-induced transformation. The implications ofthis observation are significant, as p53 integrity is crucial inminimizing tumorigenic potential in multiple tissues, includingthe mammary gland (Lozano and Liu, 1998; Jerry et al., 2000).Induction of apoptosis in response to DNA damage is a well-documented mechanism by which p53 may suppresstumorigenesis in vivo (Symonds et al., 1994). In fact, manytherapeutic strategies target the more rapidly dividing tumorcells with DNA-damaging agents as a means of eliminatingthem through p53-mediated apoptosis (Lowe et al., 1993b) andabrogation of p53 function can contribute significantly toradio- or chemoresistance (Lowe et al., 1994).

Loss of this protective mechanism may promote theaccumulation, rather than the deletion, of cells with severegenetic damage. In the small intestines, but not the colon, cellscorresponding to putative stem cells in the hierarchy of theintestinal crypts undergo apoptosis following γ-irradiation,suggesting a mechanistic basis for the decreased incidence ofradiation-induced carcinoma in the small intestines comparedto the colon (Merritt et al., 1994). Failure of irradiated virginmammary epithelium to undergo apoptosis may represent asituation comparable to that of the colon, in which putativestem cells incur DNA damage but they are not removed by p53-mediated cell death.

In addition to apoptosis, another important role of p53 ismediating repair of double strand DNA breaks following γ-irradiation. Previous studies have identified rapidlyproliferating cell populations as being highly susceptible toradiation-induced transformation (Chan and Little, 1981).However, Ullrich et al. have demonstrated increased genomicinstability in irradiated mammary epithelial cells allowed toremain in situ for a period of time following irradiation (Ullrichet al., 1999), suggesting DNA repair is compromised in thesecells. These data argue that a population of quiescent cells maybe equally susceptible to the effects of radiation-inducedgenetic instability. Furthermore, previous studies by Ponnaiyaet al. (Ponnaiya et al., 1997) have suggested a mechanistic linkbetween instability and radiation-induced cancer.

Therefore, the functional repression of p53 observed inquiescent mammary epithelial cells not only renders themresistant to apoptosis, it may also compromise their ability todetect and repair DNA damage. Though these mechanismsmay preserve the integrity of the tissues, they may also allow

increased accumulation of mutations. As such, developmentalregulation of p53 activity may represent a molecular basis fortumor susceptibility in mammary epithelial cells.

The authors would like to thank Anneke C. Blackburn and DavidA. Steele for thoughtful discussions and critical reading of themanuscript, and Sharon Marconi for expert technical assistance inprocessing mammary tissues. This work was supported by a graduatefellowship to L. M. M. from the Massachusetts Department of PublicHealth (340006670) and grants to D. J. J. from the National Institutesof Health (CA66670, CA87531) and the Massachusetts Departmentof Public Health (43088PP1017). This material is based on worksupported by the Cooperative State Research Extension, EducationService, US Department of Agriculture, Massachusetts AgriculturalExperiment Station and Department of Veterinary and AnimalSciences under Project Nos. MAS00821 and MAS00714.

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