MOLECULAR CARCINOGENESIS 22:199–207 (1998)

© 1998 WILEY-LISS, INC.

Radiation-Induced Tumorigenesis in Preneoplastic MouseMammary Glands In Vivo: Significance of p53 Status andApoptosisDaniel Medina,1* L. Clifton Stephens,2 Pedro J. Bonilla,3 C. Annette Hollmann,3 Denise Schwahn,1

Charlotte Kuperwasser,4 D. Joseph Jerry,4 Janet S. Butel,3 and Raymond E. Meyn5

1Department of Cell Biology, Baylor College of Medicine, Houston, Texas2Department of Veterinary Medicine and Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas3Department of Molecular Virology, Baylor College of Medicine, Houston, Texas4Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts5Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

In mouse mammary tumorigenesis, p53 mutations facilitate tumorigenesis in concert with other oncogenicalterations. Ionizing radiation enhances tumorigenesis in preneoplastic mammary outgrowth lines and in-duces p53-dependent apoptosis. We asked if normal p53 function modulates radiation-induced tumorigenesisin preneoplastic mammary lesions by affecting the apoptotic pathway of cell deletion. Three different hyper-plastic outgrowth lines were compared. Outgrowth line D1 overexpressed wild-type p53 and responded toirradiation with enhanced tumorigenicity but no induction of apoptosis. Outgrowth line TM12 exhibited nor-mal wild-type p53 expression and responded to irradiation with no alteration in tumorigenicity but with amarked increase in apoptosis. Outgrowth line TM2L also exhibited normal wild-type p53 expression and re-sponded to irradiation with a marked enhancement in both tumorigenicity and apoptosis. These results indi-cate that the two radiation-induced responses, apoptosis and tumorigenesis, are dissociable events in themammary gland. Furthermore, radiation-induced tumorigenicity was not abrogated by either enhanced wild-type p53 expression or a robust apoptotic response. The radiation dose of 5 Gy most likely induces multiplegenetic alterations in surviving cells, including genomic instability, and this may account for the tumorigenic-ity. Future experiments will examine lower doses of irradiation that still induce a significant apoptotic responsebut significantly less genomic instability. Mol. Carcinog. 22:199–207, 1998. © 1998 Wiley-Liss, Inc.

Key words: radiation; mouse; mammary glands; p53; apoptosis

INTRODUCTION

Breast cancer is a heterogeneous disease that mani-fests after a long latent period; both features reflectthe complex and multistage nature of the disease [1–3]. Insights into the molecular basis of breast cancerhave identified both activation of cellular proto-oncogenes [4,5] and functional loss of tumor sup-pressor genes [6–8] as important events in theprogression of this disease. The accumulation ofmultiple changes within a cell, rather than the abso-lute sequence of molecular events, appears to be thecritical factor in tumor development.

Mutations in the tumor suppressor gene p53 areamong the most common genetic changes in hu-man breast cancer, as up to 50% of cancers have al-terations in this gene [8]. One proposed primaryfunction of p53 is to safeguard the genetic integrityof the cellular genome [9,10]. Upon DNA damage,p53 can alter the cell cycle by inducing the transcrip-tion of genes involved in negative regulation of thecycle [10]. Alternatively, p53 can channel the cellsinto apoptosis [11,12]. One mechanism by which lossof normal p53 function could facilitate tumorigen-

esis is failure of genetically damaged cells to undergoapoptosis.

The mouse mammary tumor model is a paradigmfor the multiple-stage development of breast cancer.Breast cancer is influenced by multiple independentand interactive factors (viruses, genetics, hormones,chemical carcinogens, and radiation) [13]. Thepreneoplastic stages of the mouse model are welldefined and allow assessment of the relative abilitiesof specific etiological agents to disrupt proliferationcontrols at the early stage of progression [14]. Forinstance, a series of preneoplastic mouse mammarylines has been established by serial transplantationinto the cleared fat pads of syngeneic BALB/c femalemice [15]. The biological and tumorigenic proper-ties of these outgrowth lines have been well estab-

*Correspondence to: Department of Cell Biology, Baylor Collegeof Medicine, One Baylor Plaza, Houston, TX 77030.

Received 1 January 1998; Revised 11 February 1998; Accepted 13February 1998

Abbreviations: AI, apoptotic index; PCR, polymerase chain reaction

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lished [16]. The question of whether mutations inp53 play a role in the development of preneoplasiahas been addressed in this system. The results showedthat, whereas alterations in p53 genotype or expres-sion are not obligatory for establishment ofpreneoplasia, such changes are frequent and are se-lected for during the development of hyperplasticalveolar nodules, resulting in cell populations en-riched for p53 mutations [17]. Analysis of a series ofmammary preneoplasias, designated TM2H throughTM12, demonstrated that alterations in p53 proteinexpression are not obviously correlated with the abil-ity to progress from preneoplasia to overt tumors [18].Although p53 mutations confer a growth advantageto cells in vitro and in vivo and the p53 pathwaymay be a common target for mutation in murinemammary tumorigenesis, such mutations are notsufficient for mammary tumorigenesis. This conclu-sion is supported by studies using p53-null mice, inwhich spontaneous mammary tumorigenesis was notenhanced [19]. It appears rather that p53 mutationsmay facilitate carcinogenesis in concert with otherspecific oncogenic abnormalities. For example, it hasbeen demonstrated that the p53-null phenotype en-hances mammary carcinogenesis in wnt-1 transgenicmice and promotes chromosomal instability in mam-mary tumors [20].

We have used the mouse mammary tumor modelsystem to determine whether loss of normal p53 func-tion facilitates tumorigenesis in preneoplastic lesionsby abrogating the apoptotic pathway of cell deletion.In a previous study, we examined the ability of aspecific DNA-damaging agent, ionizing radiation, toinduce apoptosis in the normal mammary tissue ofBALB/c mice and compared this response to thatobserved in different preneoplastic outgrowths thathad defined p53 alterations [21]. Ionizing radiationwas chosen as the DNA-damaging agent in these stud-ies because it had been shown previously to enhancetumorigenesis in similar mammary outgrowth lines[22] and to induce p53-dependent apoptosis [11,23].Normal mammary glands and the preneoplastic out-growths with normal p53 (i.e., TM12) exhibit radia-tion-induced apoptosis. In contrast, preneoplasticoutgrowths with dysregulated p53 (i.e., D1, TM2H, andTM4) fail to demonstrate radiation-induced apoptosis.These results are consistent with the hypothesis thatnormal p53 function is important for removing byapoptosis mammary cells that harbor DNA damage.

In the results reported herein, we extended thesestudies to examine the significance of p53 andapoptosis in preneoplastic mammary gland in whichradiation-induced tumorigenesis is the endpoint.

MATERIALS AND METHODS

Mammary Outgrowth Lines

The preneoplastic hyperplastic outgrowths used inthese studies were maintained by serial transplanta-

tion into BALB/c mice. Tissues from donor mice weretransplanted into the cleared inguinal fat pads of 3-wk-old recipient mice. Descriptions of the TM hy-perplastic alveolar outgrowths have been reportedpreviously [15,16,18]. Three different hyperplasticoutgrowth lines were compared: line D1, whichoverexpresses p53, as detected by immunohis-tochemical staining, and lines TM2 and TM12, inwhich normal expression of p53 is not detectable byimmunohistochemical staining. Detailed informa-tion on the p53 status of mouse mammary preneo-plasias can be found in earlier reports [17,18,24].

Irradiation

BALB/c mice bearing preneoplastic outgrowths 11wk after transplantation were irradiated with a whole-body dose of 5 Gy by using a 137Cs animal irradiator.The outgrowths had completely filled the mammaryfat pad by 11 wk after transplantation, and cell pro-liferation was reduced to a steady-state rate comparedwith the rate observed during the preceding period,when the transplants were expanding in the mam-mary fat pad.

Three mice were killed 6 h after irradiation. Therationale for sampling the tissues 6 h after irradia-tion was based on the results of preliminary studieswe performed that demonstrated that between 1 and24 h, the peak apoptotic response occurred at 6 h.The mammary glands containing the transplantedoutgrowths were removed and prepared for histo-logical sectioning. Tissues were embedded in paraf-fin blocks from which 4-µm sections were cut andstained with hematoxylin and eosin. Apoptotic cellswere identified by morphological criteria describedand illustrated previously [25]. Apoptosis was scoredby microscopic examination at 400× of hematoxy-lin and eosin–stained sections on coded slides. Fivefields were selected randomly in each specimen, andin each field the apoptotic cells in mammary ductswere recorded as numbers per 100 nuclei scored andexpressed as a percentage (the apoptotic index (AI)).The AIs reported are therefore based on scoring 500nuclei for each specimen. The AIs given in Figures1–3 represent the average of all specimens withinthat group. Differences in AIs between the groupswere tested for statistical significance by using thetwo-sided Student’s t test. Results were consideredsignificantly different at P < 0.05. The rest of the micewere observed for 6–9 mo for tumor developmentand were palpated weekly for tumors. The glandswithout tumors were collected when the mice werekilled and were fixed, stained, and examined undera dissecting microscope for morphological alter-ations.

p53 Nucleotide Sequence Analysis

Total RNA was extracted from TM2L cells by usingRNazol B (Biotecx, Houston, TX), a modifiedChomcznski-Sacchi method [26]. The RNA was

p53 AND APOPTOSIS IN MAMMARY PRENEOPLASIA 201

treated with DNase I and then reverse transcribed in40-µL reactions containing 50 mM KCl; 10 mM Tris-HCl, pH 8.3; 5 mM MgCl2; 1 mM dNTPs; 1 U µLRNaseOUT (Life Technologies, Gaithersburg, MD);2.5 mM random hexamers; and 1 U/µL murine leu-kemia virus reverse transcriptase. The reverse tran-scription reaction mixtures were incubated at 25°Cfor 10 min, 50°C for 30 min, 99°C for 5 min and 4°Cfor 5 min. Portions of the reverse-transcribed RNAreaction mixtures, equivalent to 250 ng of total RNA,were used directly for polymerase chain reaction(PCR) in 50-µL reaction volumes containing 50 mMKCl; 10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl2; 0.001%(w/v) gelatin; 200 µM each dNTP; 0.5 µM primers;and 0.025 U/µL AmpliTaq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT). The thermal cycling pro-file used consisted of a preincubation step at 95°Cfor 220 s followed by 35 cycles consisting of DNAdenaturation at 94°C for 20 s, annealing at 61°C for20 s and extension at 72°C for 20 s. After cycling,the reaction mixtures were incubated at 72°C for 3min. All temperature incubations were performed ina GeneAmp PCR System 2400 thermal Cycler (Perkin-Elmer Corp.). Three pairs of murine p53-specific prim-ers were used to amplify the TM2L p53 gene. Thesequences of the three primer sets used were5´-GGACCATCCTGGCTGTAGGTAGCG-3´ and 5´-GCTGGCAGAATAGCTTATTGAGGG-3´ for primerset A, 5´-CCCTGTCATCTTTTGTCCCTTCTC-3´ and5´-CCCTTCTGGTCTTCAGGTAG-3´ for primer set B,and 5´-CCACTTGATGGAGAGTATTTCACCCTC-3´and 5´-GCCAGCAGAGACCTGACAACTATCAAC-3´for primer set C. In each case, the first primer listedis the forward (positive sense strand) primer and thesecond is the reverse (complementary to the posi-tive sense strand). Primer pair A was used to amplifythe p53 region between nt 113 and 616 according tothe nucleotide designation of Ozbun et al. [17].Primer pairs B and C were used to amplify the p53region between nt 484 and 1328 and nt 1171 and1583, respectively. Together these three PCR prod-ucts span a portion of the noncoding exon 1, thecoding exons 2–11, and a portion of the 3´untranslated region of the murine p53 gene. The PCRproducts were purified using a QIAquick PCR purifi-cation kit (QIAGEN Inc., Chatsworth, CA) and di-rectly sequenced with an ABI PRISM dye terminatorcycle sequencing reaction kit with AmpliTaq DNApolymerase-FS (Perkin-Elmer Corp.). For each PCRproduct the sequence of each strand was determined.For analysis of the p53 nucleotide sequence in TM12cells, we followed the methods described by Jerry etal. [18]. The primers used for PCR and direct sequenceanalysis were those described in that report.

Immunohistochemical Analysis

Paraffin-embedded sections (5 µm thick) weredeparaffinized in xylenes followed by rehydration ingraded alcohols to phosphate-buffered saline. Anti-

gen retrieval was performed in 10 mM citrate bufferin an 800-W microwave oven set on medium for 6.5min [27]. The slides were washed in phosphate-buff-ered saline and then in 0.3% hydrogen peroxide toblock endogenous peroxidase activity. Nonspecificbinding was blocked with 5% maleate buffer andMagic Blocker (Boehringer Mannheim Corp., India-napolis, IN) with 0.75% Triton X-100 followed byAvidin Blocker (Vector Labs, Inc., Burlingame, CA) for15 min each. The tissues were incubated with apolyclonal antiserum (CM5 diluted 1:200; Novacastra,Newcastle upon Tyne, UK) in a 2% maleate buffer andMagic Blocker solution for 24 h at 4°C. The complexeswere detected by the ABC method by using VectastainElite reagents (Vector Labs, Inc.) and diaminoben-zidene color substrate as suggested by the manufac-turer. Sections were counterstained with methyl green,dehydrated, and then mounted with Preservastide (EMScience, Gibbstown, NJ).

Statistics

The data were analyzed by several statistical tests.The AI was evaluated by two-sided Student’s t test,the differences in tumor incidences by the chi squareprocedure [28], and the tumor latency period by life-table procedures described by Mantel [29]. The resultswere considered significantly different at P < 0.05.

RESULTS

p53 Status in Preneoplastic Outgrowth Lines

Previous studies demonstrated that p53 protein isoverexpressed in D1 preneoplastic outgrowths andis localized in the nucleus using immunohistochemi-cal staining. However, nucleotide sequence analysisindicated that the p53 gene in those cells is wild type[24]. In the experiments described here, p53 proteinwas detectable by immunohistochemical staining inthe nuclei of less than 5% of the cells in both theTM2L and TM12 preneoplastic outgrowths (data notshown). This extent of p53 expression is consideredto be normal [24,30,31]. Nucleotide sequence analy-ses indicated that the p53 genes were wild type inTM12 (exons 4–9) and in TM2L (exons 1–11) (datanot shown).

Radiation-Induced Apoptosis and Tumorigenesis

Radiation-induced apoptosis and tumorigenesis inthe preneoplastic outgrowth lines D1, TM12, andTM2L are illustrated in Figures 1–3, respectively.Outgrowth line D1 overexpressed wild-type p53, hada very low spontaneous tumorigenic potential, andresponded to irradiation with no significant induc-tion of apoptosis but with a marked enhancementin tumor development (<10% to 44%) (Figure 1). Analmost identical response to radiation-induced tu-morigenesis was observed in early transplant gen-erations of this outgrowth line in experimentsperformed almost 30 yr ago [22]. Thus, in this line,

202 MEDINA ET AL.

dysregulated p53 wild-type expression was associatedboth with a block in radiation-induced apoptosis andwith enhanced tumorigenicity.

Outgrowth line TM12 expressed wild-type p53, hada moderate spontaneous tumor incidence and meantumor latency period, and responded to irradiationwith significantly enhanced apoptosis (1.5% to12.5%) but with only a slight, nonsignificant increasein tumorigenesis (61% to 72%) and a modest butsignificant decrease in 50% tumor endpoint (5 wk)(Figure 2). This figure is the summary of two inde-pendent experiments performed a year apart thatyielded identical results. Thus, in this line, wild-typep53 was associated with inducible apoptosis but noprotection against radiation-induced tumorigenesis.

Outgrowth line TM2L contained wild-type p53,had a low spontaneous tumor incidence, and re-sponded to irradiation with both enhanced apoptosis(2% to 11%) and tumorigenesis (20% to 61%) (Fig-ure 3). Taken together, the results for all three out-growth lines indicated that the two radiation-inducedresponses, apoptosis, and tumorigenesis, were inde-pendent events.

The mammary gland samples collected at 6 h afterirradiation were examined for p53 protein expres-sion by immunohistochemical analysis (Figure 4A–D). In irradiated TM2L and TM12 outgrowths, intensenuclear staining of p53 protein was observed at ahigh frequency (60–63%) in mammary epithelial cells

(Figure 4B and D), in contrast to the low level of dif-fuse cytoplasmic staining and infrequent nuclearstaining in the unirradiated controls (Figure 4A andC). Thus, the induction of apoptosis in these twooutgrowth lines by radiation was correlated withenhanced expression and nuclear localization of p53.However, radiation-induced tumorigenicity was notinhibited by enhanced wild-type p53 expression, asillustrated by the TM2L outgrowth line.

We subsequently examined p53 protein localiza-tion in TM12 tumors arising in control and irradi-ated mice. In tumors arising in irradiated mice,nuclear p53 protein was undetectable in 14 of 15tumors, an incidence comparable to that of control(unirradiated) mice, in which p53 protein in thenucleus was undetectable in 10 of 12 tumors.

Examination of whole mounts of the mammaryglands from non–tumor-bearing mice at 7–9 mo af-ter the initial irradiation revealed a significant lossof alveolar density in irradiated glands as comparedwith control glands (Figure 5) in all three outgrowthlines. The most extreme result was observed in lineD1, in which the entire gland was composed of veryfine ductules (Figure 5A and B), a result observed in60% of the glands. In outgrowth line TM2L, therewere variable degrees of alveolar loss in most of theglands; the typical response whereby alveoli werelost, resulting in a heterogeneous-appearing alveo-lar morphology is illustrated in Figure 5C and D.

Figure 1. Radiation-induced apoptosis and tumorigenicityin outgrowth line D1. The tumor incidence data are plotted aspercent of outgrowths developing tumors versus time after ir-radiation. The numbers in parentheses are the number of trans-

plants developing tumors over the total number of transplantsper group. The AI is plotted as percent apoptotic cells. Eachgroup was sampled in triplicate.

p53 AND APOPTOSIS IN MAMMARY PRENEOPLASIA 203

Outgrowth line TM12 lost the fewest alveoli of thosethree outgrowth lines and lost alveoli in only 20%of the outgrowths (Figure 5E and F). Thus, there wasno clear association between radiation-induced lossof alveolar morphology and induction of apoptosis,indicating that the loss of alveolar cells was due to amechanism other than apoptosis.

DISCUSSION

Normal p53 function is required for apoptosis toproceed after DNA damage [12,23]. This idea is sup-ported by results in transgenic mice in which thy-mocytes lacking p53 do not undergo radiation-inducedapoptosis but do undergo apoptosis in response to glu-cocorticoids [12,23]. Similarly, radiation-inducedapoptosis is blocked in the gastrointestinal tract ofp53-null mice [32] and in mammary preneoplasiaswith no or mutant p53 [21]. Based on these resultsand the currently accepted concept that p53 protectstissues from genetic damage by deleting the affectedcells by apoptosis [10,12,33], we designed experi-ments to determine whether abrogation of apoptosisenhances radiation-induced tumorigenesis.

It is evident from the results reported herein thatradiation-induced apoptosis and tumorigenicity were

not necessarily interrelated and predictable from thep53 status of the preneoplastic outgrowths (Table 1).There was clearly a tight correlation between p53 sta-tus and radiation-induced apoptosis in the fivepreneoplastic outgrowth lines examined in this andour previous study [21]. Apoptosis was increased onlyin the presence of normally regulated wild-type p53.However, the enhancement of tumorigenesis in theTM2L and, to a much lesser extent, in the TM12outgrowth line suggests that a more complex arrayof interactions (e.g., induction of genomic instabil-ity, response to DNA damage, and alteration in geneexpression) determines the tumorigenic response andthat at least in TM2L, a robust apoptotic response isnot sufficient to protect irradiated tissues from tu-mor development.

Several considerations may be important in un-derstanding the relationship between apoptosis andtumor development in irradiated preneoplastic le-sions. The single dose of 5 Gy is a relatively highdose of radiation, and one would predict significantgenetic alterations in cells surviving such a treatment.It is conceivable that such genetic alterations wouldresult in a dysregulation of cell proliferation and in-vasiveness that would offset any removal of cells by

Figure 2. Radiation-induced apoptosis and tumorigenicity in outgrowth line TM12. The data areplotted as in Figure 1.

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Figure 3. Radiation-induced apoptosis and tumorigenicity in outgrowth line TM2L. The dataare plotted as in Figure 1.

Figure 4. p53 protein expression detected by immunohis-tochemical analysis in irradiated and unirradiated outgrowthsexamined at 6 h after irradiation. Note the intense nuclear stain-ing of p53 protein in irradiated specimens compared with the

weak cytoplasmic reactivity in unirradiated control specimens.(A) Unirradiated TM12; (B) irradiated TM12; (C) unirradiatedTM2L; (D) irradiated TM2L. Magnification, 120×

p53 AND APOPTOSIS IN MAMMARY PRENEOPLASIA 205

Figure 5. Whole mounts of control and irradiated mam-mary outgrowths in non–tumor-bearing mice. The mammaryoutgrowths were collected in age-matched animals 7–9 mo afterirradiation. Each outgrowth line exhibits some degree of al-

veolar cell loss in the irradiated glands. (A) Unirradiated D1;(B) irradiated D1; (C) unirradiated TM12; (D) irradiated TM12;(E) unirradiated TM2L; (F) irradiated TM2L. Magnification, 6×

apoptosis. This interpretation is supported by theanalysis of outgrowth morphology after irradiation.Both the TM12 and TM2L outgrowth lines exhibitedsimilar degrees of apoptosis; however, alveolar cellloss was not correlated with the radiation-inducedapoptosis, which suggests that irradiation had othereffects on cell growth and maintenance. Regardlessof the precise mechanism of cell loss, both TM2L

and D1 exhibited marked enhancement of tumori-genesis in response to irradiation. Thus, it seemslikely that preexisting genetic alterations in TM2Lcooperated with irradiation-induced alterations toproduce a tumorigenic response that more than com-pensated for any apoptosis-related cell loss. It is sig-nificant that mammary tumors never arose inirradiated thoracic (normal) mammary glands in the

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same animals. At this time, we do not have enoughinformation on radiation-induced genetic alterationsto identify their precise mechanisms. However, it isimportant to consider the emerging data suggestingthat ionizing radiation is an extremely potent agentfor inducing genomic instability [34]. With regardto radiation-induced mammary cancer, Ullrich andcoworkers [35] reported that the neoplastic changesarising from irradiated mouse mammary tissue aremost likely the result primarily of the induction ofgenomic instability, with alterations in p53 and othergenes being secondary consequences of this ge-nomic instability. Their data [35] indicated thatthe induction of genomic instability by irradia-tion in mammary epithelial cell cultures from twostrains of mice sensitive and resistant to radiation-induced mammary cancer directly correlates withthe strain’s susceptibility to mammary-tumor in-duction. The data presented here are entirely con-sistent with this model.

It has been reported that p53-deficient mice areextremely susceptible to radiation-induced tumori-genesis, with the tumors being predominantly lym-phomas and soft-tissue sarcomas [36]. However, p53in cells is either normal or functionally mutant ratherthan deleted. In our model system, we could notexamine the effects of either a dominant negativemutant of p53 (TM4) or the p53-null phenotype(TM2H), as these outgrowth lines had a high rate ofspontaneous tumorigenicity. However, we did com-pare an overexpressed wild-type p53 phenotype withthe normal p53 phenotype. The results indicated thatthe tumorigenic response to irradiation was not pre-dictable by either p53 status or apoptotic response.This model system appears to be useful for dissect-ing the relative contribution of apoptosis to coun-teracting tumorigenesis. However, examination oflower doses of radiation is warranted, as 5 Gy is arelatively high dose and so induces considerable tox-icity. It would be of interest, therefore, to evaluatethe response of these outgrowths at doses of 0.5 Gyor less, because of a report indicating that genomicinstability is less after a low dose of radiation [35]and because we have shown that 0.5 Gy induces sub-stantial apoptosis in mouse mammary cells (MeynRE, unpublished results).

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistanceof Frances Kittrell in performing the transplantationexperiments and Elizabeth Hopkins for preparationof histological sections. These studies were supportedby CA 25215 (to JB and DM), CA 06294 and CA69003(to REM), and CA66670 (to DJJ).

REFERENCES1. Kelsey JL, Gammon MD. The epidemiology of breast cancer. CA

Cancer J Clin 41:146–165, 1991.2. Allred DC. Biological and genetic features of in situ breast can-

cer. In: Silverstein MJ (ed), Ductal Carcinoma In Situ of the Breast.Williams and Wilkins, Baltimore, 1997, pp. 37–49.

3. Varmus HE, Godley LA, Roy S, et al. Defining the steps in a multi-step mouse model for mammary carcinogenesis. Cold SpringHarb Symp Quant Biol 59:491–500, 1995.

4. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuireWL. Human breast cancer: Correlation of relapse and survivalwith amplification of the HER2/neu oncogene. Science 235:177–182, 1987.

5. DairKee SH, Smith HS. Genetic analysis of breast cancer progres-sion. Journal of Mammary Gland Biology and Neoplasia 1:139–151, 1996.

6. Gayther SA, Warren W, Mazoyer S, et al. Germline mutations ofthe BRCA1 gene in breast and ovarian cancer families provideevidence for a genotype-phenotype correlation. Nat Genet11:428–433, 1995.

7. Asch HL, Head K, Dong Y, et al. Widespread loss of gelsolin inbreast cancers in humans, mice, and rats. Cancer Res 56:4841–4845, 1996.

8. Ozbun MA, Butel JS. Tumor suppressor p53 mutations and breastcancer: A critical analysis. Adv Cancer Res 66:71–141, 1995.

9. Lane D. p53, guardian of the genome. Nature 358:15–16, 1992.10. Kuerbitz S, Plunkett B, Walsh W, Kastan M. Wild-type p53 is a

cell cycle checkpoint determinant following radiation. Nature89:7491–7495, 1992.

11. Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, OrenM. Wild-type p53 induces apoptosis of myeloid leukaemic cellsthat is inhibited by interleukin-6. Nature 352:345–347, 1991.

12. Lowe S, Schmit E, Smith S, Osborne B, Jacks T. p53 is requiredfor radiation-induced apoptosis in mouse thymocytes. Nature362:847–849, 1993.

13. Medina D. Mammary tumors in mice. In: Foster HL, Small JD, FoxJG (eds), The Mouse in Biomedical Research, Vol 4. AcademicPress, New York, 1982, pp. 373–396.

14. Medina D. Preneoplasia in mammary tumorigenesis. In: DicksonRB, Lippman ME (eds), Mammary Tumor Cell Cycle, Differentia-tion and Metastases. Kluwer Academic Publishers, Norwell, MA,1996, pp. 37–69.

15. Kittrell F, Osborn C, Medina D. Development of mammarypreneoplasias in vivo from mouse mammary epithelial cell linesin vitro. Cancer Res 52:1924–1932, 1992.

16. Medina D, Kittrell F, Liu Y-J, Schwartz M. Morphological andfunctional properties of TM preneoplastic mammary outgrowths.Cancer Res 53:663–667, 1993.

17. Ozbun M, Jerry D, Kittrell F, Medina D, Butel J. p53 muta-tions selected in vivo when mouse mammary epithelial cellsform hyperplastic outgrowths are not necessary for estab-lishment of mammary cell lines in vitro. Cancer Res 53:1646–1652, 1993.

Table 1. Dissociation of Radiation-Induced Apoptosis and Tumorigenicity in Mouse Mammary Preneoplasias

Radiation-Outgrowth p53 p53 Protein Radiation-induced* enhancedline Genotype expression† Apoptosis Alveolar cell loss tumorigenicity*

D1 Wild type Elevated – + +TM2L Wild type Normal + + +TM12 Wild type Normal + ± –

†In control , unirradiated mammary outgrowths, as determined by immunohistochemical staining.*–, not significantly altered; +, significantly altered from unirradiated samples.

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18. Jerry D, Ozbun M, Kittrell F, Lane D, Medina D, Butel J. Muta-tions in p53 are frequent in the preneoplastic stage of mousemammary tumor development. Cancer Res 53:3374–3381, 1993.

19. Donehower L, Harvey M, Slagle B, et al. Mice deficient for p53are developmentally normal but susceptible to spontaneous tu-mors. Nature 356:215–221, 1992.

20. Donehower L, Godley L, Aldaz C, et al. Deficiency of p53 accel-erates mammary tumorigenesis in wnt-1 transgenic mice andpromotes chromosomal instability. Genes Dev 9:882–895, 1995.

21. Meyn RE, Stephens LC, Mason KA, Medina D. Radiation-inducedapoptosis in normal and pre-neoplastic mammary glands in vivo:Significance of gland differentiation and p53 status. Int J Cancer65:466–472, 1996.

22. Medina D. Preneoplastic lesions in mouse mammary tumorigen-esis. Methods in Cancer Research 7:3–53, 1973.

23. Clarke A, Purdie C, Harrison D, et al. Thymocyte apoptosis in-duced by p53-dependent and independent pathways. Nature362:849–852, 1993.

24. Jerry DJ, Butel JS, Donehower LA, et al. Infrequent p53 muta-tions in 7,12-dimethylbenzanthracene–induced mammary tumorsin BALB/c and p53 hemizygous mice. Mol Carcinog 9:175–183,1994.

25. Stephens L, Schultheiss T, Small S, Ang K, Peters L. Response ofparotid organ culture to radiation. Radiat Res 120:140–153,1989.

26. Chomcznski P, Sacchi N. Single step method of RNA isolation byguanidium thiocyanate-phenol-chloroform extraction. AnalBiochem 162:156–159, 1987.

27. MacCallum DE, Hupp TR, Midgely CA, et al. The p53 response

to ionising radiation in adult and developing murine tissues.Oncogene 13:2575–2587, 1996.

28. Peto R. Guidelines in the analysis of tumor rates and death ratesin experimental animals. Br J Cancer 29:101–105, 1974.

29. Mantel N. Evaluation of survival data and two new rank orderstatistics arising in its consideration. Cancer Chemotherapy Re-ports 50:163–170, 1966.

30. MacGrogan G, Bonichon F, de Mascarel I, et al. Prognostic valueof p53 in breast invasive ductal carcinoma: An immunohis-tochemical study on 942 cases. Breast Cancer Res Treat 36:71–81, 1995.

31. Muss HB, Thor AD, Berry DA, et al. c-erbB-2 expression and re-sponse to adjuvant therapy in women with node-positive earlybreast cancer. N Engl J Med 330:1260–1266, 1994.

32. Merritt A, Potten C, Kemp C, et al. The role of p53 in spon-taneous and radiation-induced apoptosis in the gastrointes-tinal tract of normal and p53-deficient mice. Cancer Res54:614–617, 1994.

33. Arends MJ, Wyllie AH. Apoptosis: Mechanisms and roles in pa-thology. Int Rev Exp Pathol 32:223–254, 1991.

34. Morgan WF, Day JP, Kaplan MI, McGhee EM, Limoli CL. Ge-nomic instability induced by ionizing radiation. Radiat Res146:247–258, 1996.

35. Ponnaiya B, Cornforth MN, Ullrich RL. Radiation-induced chro-mosomal instability in BALB/c and C57BL/6 mice: The differenceis as clear as black and white. Radiat Res 147:121–125, 1997.

36. Kemp C, Wheldon T, Balmain A. p53-deficient mice are extremelysusceptible to radiation-induced tumorigenesis. Nat Genet 8:66–69, 1994.