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Abnormal Expression of the ATM and TP53 Genes in SporadicBreast Carcinomas1

Sandra Angèle, Isabelle Treilleux,Philippe Tanière, Ghyslaine Martel-Planche,Michèle Vuillaume, Christiane Bailly,Alain Brémond, Ruggero Montesano, andJanet Hall2

DNA Repair Group [S. A., M. V., R. M., J. H.] and MolecularCarcinogenesis Group [P. T., G. M-P., R. M.], International Agencyfor Research on Cancer, and Centre Régional Le´on Bérard [I. T.,C. B., A. B.], 69372 Lyon Cedex 08, France.

ABSTRACTThe ataxia telangiectasia gene (ATM) has been impli-

cated as a risk factor in the development of sporadic breastcarcinomas. ATM protein expression was analyzed by im-munohistochemistry in 17 breast carcinomas with twomonoclonal antibodies whose immunohistochemical use wasfirst validated by comparing the immunoreactivity observedin spleen samples from ataxia telangiectasia and traumapatients. In normal breast ducts, ATM showed nuclear ex-pression in the epithelial but not in the myoepithelial cells. Incontrast, this nuclear expression was absent or low in theepithelial cancer cells in 10 of 17 (59%) of the tumorsstudied. Allelic imbalance in the ATM gene was found inthree of seven tumors examined. Two of these showed re-duced ATM protein expression, but this did not correlatewith the presence of ATM mutations in the tumor DNAdetected by restriction endonuclease fingerprinting screen-ing. These results suggest that the reduced ATM proteinexpression could be attributable, in certain tumors, to dele-tions or rearrangements within or close to theATM gene.Positive p53 immunostaining was found in 10 tumors, withTP53 mutations detected in 8. Three tumors had both lowATM expression and mutated TP53. Our results indicatethat in the majority (15 of 17) of the sporadic breast carci-nomas examined, not only is the functionality of the ATM-p53-mediated DNA damage response compromised, but alsoother signaling pathways activated by these two multifunc-

tional proteins are likely to be impaired, which could be acontributing factor to tumor development and progression.

INTRODUCTIONSeveral genes are known to predispose women to breast

cancer, which is a common disease with a complex etiology.Although mutations inBRCA1and BRCA2are recognized asrisk factors for inherited breast cancer, somatic mutations inthese genes are rare in sporadic breast cancers (1, 2). In princi-ple, a greater proportion of breast cancer cases within thepopulation could be attributed to genes that are more frequentlymutated but which may have a relatively low penetrance withrespect to breast cancer. The AT3 gene (ATM) is one candidatefor such a susceptibility gene.

AT is characterized by cerebellar ataxia, skin, and oculartelangiectasias, immunodeficiency, extreme cellular sensitivityto ionizing radiation, and predisposition to cancer. Epidemio-logical studies on AT families have shown that AT heterozy-gotes also have an increased risk of developing cancer, inparticular breast cancer, for which femaleATM carriers have a4-fold increased risk compared with the general population(3–5). It has been estimated that because;1% of the generalpopulation are AT heterozygotes, alterations in theATM genecould account for up to 8% of all breast cancer cases. A role forthe ATM gene in sporadic breast cancer is supported by manystudies that have shown a LOH in the region of theATM genelocated on chromosome 11q23.1. This has been found in;40%of tumors studied (6–10). A causative association with theATMgene has, however, been shown in only a few familial cases ofbreast cancer (11–13), and to date, fewATM mutations havebeen reported in sporadic or early onset breast cancer (for arecent review, see Ref. 14).

Normal breast tissue shows a distinct pattern of ATMexpression, the protein being found in the ductal epithelial cellsbut not in the surrounding myoepithelial cells (15). In contrast,in cases of sclerosing adenosis, a benign breast lesion, ATM isexpressed in both the epithelial and myoepithelial cells. Thisup-regulation of ATM expression was associated with prolifer-ation of the myoepithelial cells (15). Recently, Kairouzet al.(16) have reported a reduction in the level of ATM protein insporadic breast tumors.ATM mRNA levels have also beenfound to be lower in invasive breast carcinomas than in normalbreast tissue or benign lesions. This reduction was observed inbreast tumors with or without LOH in the region of theATMgene, suggesting that genetic events other than gene deletionscould result in reducedATM gene expression (17).

Received 4/3/00; revised 6/8/00; accepted 6/12/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by grants from the “Association pour la Recherchesur le Cancer” and “La Ligue Nationale Contre le Cancer, ComitéDépartemental du Rhône.”2 To whom requests for reprints should be addressed, at DNA RepairGroup, International Agency for Research on Cancer, 150 cours AlbertThomas, 69372 Lyon Cedex 08, France. Phone: 33-472738596; Fax:33-472738322; E-mail: [email protected].

3 The abbreviations used are: AT, ataxia telangiectasia; LOH, loss ofheterozygosity; TTGE, temporal temperature gradient gel electrophore-sis; IDC, invasive ductal carcinoma; REF, restriction endonucleasefingerprinting.

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Another gene in which mutations have been shown topredispose to breast cancer, especially associated with the Li-Fraumeni syndrome, is theTP53 gene. Using immunohisto-chemical approaches, elevated levels of the p53 protein havebeen found in a high percentage (20–55%) of breast tumors.However, the prevalence ofTP53 mutations in such sporadicbreast tumors is lower (15–46%), suggesting that in some tu-mors, it is wild-type p53 and not mutant protein that is beingdetected (18, 19). This apparent increase in protein expressionmay be a response to a variety of DNA stresses, including DNAdamage, oxidative stress, or hypoxia.

The interaction between the ATM and p53 proteins isessential in the cellular response to DNA damage and in partic-ular, double-strand breaks that can arise endogenously duringcellular processes, such as V(D)J recombination or meiosis, orafter exposure to DNA-damaging agents, such as ionizing radi-ation. The signaling cascades that are activated in response tosuch damage remain to be fully elucidated, but both proteinsplay critical roles. In response to ionizing radiation, the kinaseactivity of ATM is enhanced, leading to phosphorylation of p53on serine 15. The activated ATM kinase also phosphorylates andactivates the checkpoint kinase Chk2, which in turn, phospho-rylates p53 on serine 20 (20). These interactions result in thestabilization of p53 and its activation as a transcription factor ofgenes such asWAF1/Cip1,MDM2, GADD45,BAX, and IGF-BP3. Their transcriptional activation is associated with cellcycle arrest, DNA repair, or apoptosis (21). ATM can alsoregulate the cell cycle by p53-independent pathways, involvingc-Abl, replication protein A, and Chk2 (21, 22). Both of thesemultifunctional proteins are also involved in various other sig-naling pathways (23, 24). Thus, abnormalities in either ATM orp53 expression could have dramatic consequences for both the

control of normal physiological processes and the cellular re-sponse to DNA damage. The aim of the present study was toinvestigate whether there was a correlation between the proteinexpression profiles of ATM and p53 and the mutational status oftheTP53gene in breast carcinomas using immunohistochemicaltechniques and DNA analysis.

MATERIALS AND METHODSPatient Samples. This study was approved by the local

ethical committees. Breast tissue was obtained from 17 un-selected female patients undergoing surgery for breast cancer atthe Centre Régional Le´on Bérard (Lyon, France). Sixteen sam-ples were IDCs according to WHO criteria. Two of these tumorswere associated with anin situ ductal carcinoma. One samplewas exclusively anin situ ductal carcinoma. The grade of theinvasive carcinomas was evaluated according to Scarff-Bloom-Richardson grading (25). None of the patients was treated withradiotherapy, but three patients had received treatment beforesurgery: one with chemotherapy, one with hormone therapy, andone with both (see Table 1). Six non-cancer (normal) breastsamples from reduction mammoplasties were used as controls.All samples were snap-frozen in liquid nitrogen and stored at270°C until further processing. Two formalin-fixed paraffin-embedded spleen samples from clinically diagnosed AT patientsand two age-matched spleen samples from trauma patients wererespectively used as negative and positive controls for the ATMstaining.

Immunohistochemical Analysis for ATM and p53 Ex-pression. Frozen tissues were directly fixed in 4% formalinfor 12–24 h and then paraffin-embedded. For each sample,morphological assessment was carried out on a 4-mm tissue

Table 1 ATM immunostaining andTP53status in breast carcinomas

Sample no. Histology Grade

ATM expression

TP53status

Immunostaining Mutationsd

Intensity % positive cells CM1 DO-7 Codon Exon Base AA change

TS1a IDC III Low 10 2 2 NoneTS2 IDC III High 10 1 1 174 del 18 5 AGG3GAG Arg3GluTS3 IDC III Low 50 1 1 NoneTS4 IDC I High .80 1 2 NoneTS5 IDC II Moderate to high .80 2 2 NoneTS6 IDCc III Low to moderate 80 1 1 248 7 CGG3TGG Arg3TrpTS7 IDC II Low .80 2 2 NoneTS8 IDC II Low 20–50 1 1 248 7 CGG3TGG Arg3TrpTS9 IDC II Moderate 50 1 1 273 8 CGT3CTT Arg3LeuTS10b IDC III Moderate to high .80 1 1 248 7 CGG3TGG Arg3TrpTS11 IDCc III Low to moderate 20–50 1 1 248 7 CGG3CAG Arg3GlnTS12 IDC II 0 0 2 2 NoneTS13 DCISe Low 20 2 2 NoneTS14 IDC II 0 0 2 2 NoneTS15a,b IDC II Low 10–20 1 1 279 8 GGG3GAG Gly3GluTS16 IDC III Low 50–80 1 1 248 7 CGG3CAG Arg3GlnTS17 IDC II Low 20 2 2 Nonea Patients received hormone therapy before surgery.b Patients received chemotherapy before surgery.c Associated within situ carcinoma.d Exons 4–9 screened.e DCIS, ductal carcinomain situ.

3537Clinical Cancer Research



section stained with H&E, and the following sections were usedfor immunostaining. All tumor samples contained.80% tumorcells. The heat-induced epitope retrieval was optimized for boththe spleen and breast tissues such that a distinct signal could bedetected in the lymphocytes, which served as an internal posi-tive control. After deparaffinizing, the endogenous peroxidaseswere inactivated by incubation for 30 min in 0.3% H2O2/methanol, and the slides were rehydrated. For ATM analysis,each section was then treated with antigen unmasking solution(Vector Laboratories Inc., Biosys S.A., Compiègne, France)either for three times for 5 min each in a microwave oven for thebreast tissues or by pressure cooking for 10 min for the spleentissues. The slides were allowed to cool in this solution andsubsequently washed in PBS (three times for 5 min each).Antigen demasking was not necessary for p53 analysis. Afterblocking the nonspecific protein binding with PBS-5% skimmedmilk-0.1% BSA for 45 min, the slides were incubated overnightat 4°C with the primary antibody. For ATM detection, twodifferent monoclonal antibodies were used: ATM1 (ATM132)or ATM2 (ATML2; 1/20 dilutions) raised against amino acids819–844 and 2581–2599, respectively (Ref. 26; generouslyprovided by Dr. Yosef Shiloh, Tel Aviv University, RamatAviv, Israel). For p53 detection, the polyclonal CM1 antibody(1/500 dilution; Novocastra Laboratories Ltd, Newcastle,United Kingdom) and the monoclonal DO-7 antibody (1/50dilution; Dako S.A., Trappes, France), which both recognizewild-type and mutant forms of the human p53 protein, wereused. All of the antibodies were diluted in PBS-0.1% BSA.After PBS washings (three times for 5 min each), slides wereincubated for 45 min with the secondary antibody: an antimouse(ATM1, ATM2, and DO-7) or an antirabbit (CM1) biotinylatedserum (Vectastain ABC Kit, Vector Laboratories Inc.; 1/200dilution in PBS-0.1%BSA). The sites of peroxidase binding wererevealed by diaminobenzidine staining (Vector Laboratories Inc.)after streptavidin signal amplification (Vectastain ABC Kit,Vector Laboratories Inc.). Mayer’s hematoxylin was used forcounterstaining before dehydration and mounting of slides. Insections used as negative controls, the primary antibodies wereomitted. The cellular localization, intensity, and the percentageof cells with positive ATM staining were assessed on the wholesection by two investigators. In the case of p53, tumors wereconsidered as immunohistochemically positive when.10% ofthe tumor cells showed nuclear p53 staining.

DNA Extraction and Microsatellite Analysis. Forseven tumors, DNA could be extracted from both the tumor andthe surrounding noninvolved normal tissue by microdissection.For each tissue sample, four 5-mm sections were deparaffinized,and the appropriate areas of tissue were incubated at 55°C in 50ml of TE buffer [10 mM Tris, 1 mM EDTA (pH 9.0)] containing0.1 mg/ml proteinase K and 0.25% NP40. Proteinase K, at thesame concentration, was added every 12 h until tissue digestionwas complete and then inactivated by heating at 95°C for 10min. Samples were centrifuged for 2 min at 10,000 rpm, and thesupernatant was stored at 4°C.

Allelic imbalance was determined at theATM locus usingtwo intragenic microsatellite markers,D11S2179and NS22, andthree closely flanking markers,D11S1819,D11S1294,andD11S1818. Primer sequences and PCR conditions for the NS22marker (ATM7F and NS22R primers) were those of Udaret al.

(27) and for all of the other microsatellite markers as describedin the Genome Database.4 One 59-nucleotide of each primer pairwas labeled with a different fluorescent dye. For each sample,PCR products obtained for the five different microsatellitemarkers were mixed and denatured at 95°C for 2 min. Electro-phoresis was carried out on a 4.8% denaturing polyacrylamidegel in the presence of 0.5ml of a TAMRA-labeled size standard(Genescan-500 TAMRA, Applied Biosystems, Courtaboeuf,France) in a model 377 DNA Sequencer (Applied Biosystems)for 4 h at 1000 V. All samples were processed at least twice.Collected data were analyzed using the Genescan Analysis 2.1software. A difference in the allele ratios in tumor DNA com-pared with normal tissue DNA$30% was scored as allelicimbalance, which could thus correspond to a gain or a loss ofone allele.

TP53 Mutation Detection. The TP53 mutational statusof all samples was analyzed by TTGE, which can be used toscreen DNA fragments for small sequence changes or pointmutations. DNA was extracted from 25 mg of frozen tissueusing the DNeasy Tissue Kit (Qiagen S.A., Courtaboeuf,France). To avoid any possible cross-contamination, each frozentissue sample was cut and weighed separately using disposable,sterile scalpels and dishes. Gloves were changed between han-dling each tissue sample. Exons 4–9 of theTP53 gene wereamplified using primers with a GC-rich sequence as describedby Hamelinet al. (Ref. 28; exons 5–8) and Guldberget al. (Ref.29; exons 4 and 9). Heteroduplex formation was induced by a10-min incubation at 98°C and then 30 min at 55°C or 62°C(depending on the exon being screened). Samples were electro-phoresed in a 7.5 or 9% polyacrylamide gel containing 7 or 8Murea, respectively, in 1.253 TAE [50 mM Tris acetate, 25 mMsodium acetate, 1.25 mM EDTA (pH 7.4)]. Migration was car-ried out at 130 V with a temperature range between 53°C and70°C and a rate of change of temperature of 2°C or 3°C/haccording to the exon being analyzed.5 Samples with aberrantlymigrating bands were reamplified and a second TTGE wasperformed. Mutant alleles were cut from this second gel andreamplified using the appropriate primers. For direct sequencingof this PCR product, asymmetric PCR amplifications were per-formed as previously described (30). Reactions were run onpolyacrylamide-urea gels, dried, and exposed to Kodak BioMaxMR-1 autoradiography films. The mutation spectra found werecompared with those reported in the IARCTP53 mutationdatabase.6

RESULTSImmunohistochemical Characterization of ATM Ex-

pression. The two ATM monoclonal antibodies used in thisstudy each recognize aMr 370,000 protein on Western blots ofprotein extracts prepared from normal cells and AT cells ectopi-

4 Internet address: http://www.gdb.org/gdb/.5 P. Tanière, G. Martel-Planche, D. Maurici, C. Lombard-Bohas, J-Y.Scoazec, R. Montesano, F. Berger, and P. Hainaut. Tumors of theesophago-gastric junction: molecular and clinical differences betweenadenocarcinomas of the esophagus and of the gastric cardia, submittedfor publication.6 Internet address: http://www.iarc.fr/p53/homepage.htm.

3538ATM andTP53 in Sporadic Breast Carcinomas



cally expressing ATM, but this protein is absent in AT cells(26). The comparison of the staining pattern in tissue sectionsfrom two AT patients and two control subjects validated the useof these two antibodies for immunohistochemical detection ofATM in tissue sections: both antibodies gave identical results.The lymphocytes in the spleens of the control subjects showedpositive ATM immunostaining (Fig. 1,a and c) but not thelymphocytes in either of the tissue sections from the AT patients(Fig. 1, b andd). Although the exact nature of theATM muta-tions in these AT patients is not known, they showed all of thecharacteristic clinical symptoms of the disease.7 The vast ma-jority of such patients have null mutations in theATM gene,which are predicted to give rise to a truncated protein that isunstable and not detected by immunological techniques (31).

In the six normal breast tissue samples examined, the ATMprotein was detected in the nucleus of the epithelial cells but wasabsent in the ductal myoepithelial cells (Fig. 2,a--c). Thisprofile is identical to that previously reported for normal breasttissue (15, 16). In contrast, in 56% of the IDCs examined (9 of16), the nuclear ATM staining in the epithelial cancer cells wasabsent or at a low intensity compared with that seen in thenormal epithelial breast cells (Fig. 2,e--gand Table 1). Seven of17 tumors expressed ATM protein at moderate to high levels. Insome cases, the ATM expression was similar to that seen in nor-mal epithelial cells: these latter cases are graded as “high” inTable 1. Two of 17 breast tumors exhibited no detectable ATMexpression, although ATM expression could be detected in thelymphocytes within the same tissue section. The only tissuesample, which was exclusively anin situ carcinoma, showed a

low intensity of nuclear ATM staining in;20% of the tumorepithelial cells. In the two cases in whichin situ carcinoma waspresent together with the invasive component, ATM expressionwas identical in both lesions.

The percentage of epithelial cells expressing ATM proteinwas variable between tumors. However, even in those sectionswhere a high percentage of the cells expressed ATM, there wasvery little inter-cell variation in the intensity of staining (Fig. 3and Table 1). In six of 17 tumors, general cytoplasmic ATMstaining was observed. This was also present in the negativecontrol for each of these six samples where no primary antibodywas used; thus, this cytoplasmic ATM staining must be consid-ered as nonspecific (data not presented).

Allelic Imbalance in the Region of the ATM Gene.Seven of 17 primary breast carcinomas from which noninvolvedand tumor tissue could be isolated by microdissection wereselected to study allelic imbalance in the region of theATMgene. The NS22 marker, which was previously shown to be ahighly informative marker in the general population (27), wasuninformative in six of seven cases examined in this study.Three breast tumors (TS5, TS8, and TS13) demonstrated allelicimbalance at theD11S2179locus located within theATM gene,and this was associated with allelic imbalance of a flankingmarker, either centromeric (D11S1819) or telomeric (D11S1294andDS11S1818). This allelic imbalance correlated in TS8 andTS13 with a low level of ATM protein expression in;20% ofthe tumor cells and thus could be considered as an allelic loss.In contrast, in TS5, a moderate to high ATM staining in.80%of the tumor cells was observed, suggesting a gene amplificationor rearrangement. The entireATM coding sequence of TS5,TS8, and TS13 was analyzed for the presence of mutations usingthe REF technique (32, 33), and noATM mutations were de-tected (data not presented). In the other four samples, there was7 S. Becker-Catania, personal communication.

Fig. 1 ATM immunostainingin spleen tissue from normaland AT patients.Left panels(magnification,3400), positiveATM immunoreactivity was ob-served using either the ATM1 (a)or the ATM2 (c) antibodies in thenucleus of the lymphocytes pres-ent in the spleen sections fromtrauma victims. Right panels(magnification,3400), no ATMexpression was detected in thespleen sections from AT patientsusing either the ATM1 (b) or theATM2 antibodies (d).




no clear correlation between ATM expression and allelic imbal-ance in the region of theATM gene.

Immunohistochemical Characterization of p53 Expres-sion. Positive nuclear p53 immunostaining was detected in 9of 16 (56%) of the IDCs examined using the monoclonal anti-body DO-7. One additional sample showed positive nuclearstaining in;20% of the tumor cells when the polyclonal anti-body CM1 was used (Table 1). Cytoplasmic staining was notobserved in this series of samples. Among the 10 tumors exhib-iting no detectable or a low level of ATM staining, six showeda negative nuclear p53 staining and four had positive nuclearp53 staining (Fig. 2h) when compared with the normal tissue(Fig. 2d).

TP53 Gene Mutations. Exons 4–9 of theTP53 genewere analyzed using TTGE. Eight of 10 tumors with positivep53 immunostaining contained aTP53 mutation (Table 1).Seven of these mutations were missense mutations, and themajority were located in exons 7 and 8. Three tumors contained

the same point mutation,CGG3TGG (Arg3Trp) at codon 248in exon 7. A different point mutation at the same codon was alsoobserved in two other tumors (CGG3CAG, Arg3Gln). Thesemutations at codon 248 were reconfirmed using a second DNAsample extracted independently from each of the five tumorsamples. One tumor contained an in-frame 18-nucleotide dele-tion at codon 174 in exon 5. No mutations were detected in theseven tumors showing negative p53 immunoreactivity with theCM1 and/or DO-7 antibodies.

DISCUSSIONUsing the immunohistochemical technique, we have dem-

onstrated a specific ATM expression profile in normal breasttissue with nuclear expression in the duct epithelial cells, whichis absent in the myoepithelial cells. Using two ATM antibodies,which recognize different epitopes on the ATM protein, wefound a significant reduction in the intensity of the nuclear ATMstaining in the epithelial cancer cells in 59% of the breast tumorsexamined. These results are in agreement with the recent reportby Kairouzet al. (16), who showed a reduction or absence ofATM protein immunoreactivity in 14 of 42 invasive breastductal carcinomas examined. Interestingly, they also noted thatthe levels of ATM were greatly reduced or absent in mostmetastatic breast carcinomas in lymph nodes (10 of 14), com-pared with the levels observed in nonmetastatic invasive breastcarcinomas. A consistent trend was found toward weaker ATMimmunostaining for more invasive stages between patients andamong all patients with progressive stages of breast cancer. Thisobservation may in part explain the higher percentage of IDCswith reduced ATM expression seen in our study because 7 of 16examined were grade III. This reduced ATM expression inbreast carcinomas is also consistent with the decrease ofATMmRNA transcript levels in breast carcinomas compared withbenign lesions and normal breast tissue reported by Wahaet al.(17).

Although the different studies have found a similar cellularprofile of ATM expression in normal breast tissue and a reduc-tion in its expression in some breast tumors, significant differ-ences have been noted in the extent of cytoplasmic stainingfound both in normal and breast cancer cells. In the presentstudy, cytoplasmic ATM immunostaining was noted in 6 of 17tumors but not, in those cases where it could be examined, in thesurrounding noninvolved epithelial cells or in any of the normalbreast tissues examined. In all of these six tumors, this cyto-plasmic staining pattern was still observed in the tumor whenthe primary antibody was omitted, but the lymphocytes, whichserved as internal positive controls for ATM expression, showedno staining under these conditions. These observations stronglysuggest that the cytoplasmic staining seen in some tumor tissueswas nonspecific. However, in the study by Kairouzet al. (16),strong immunoreactivity was seen using antibody ATM-4BA inthe cytoplasm of cells within the inner epithelial layer of normalducts in the non-neoplastic normal-appearing breast tissue in 29of 36 samples, whereas 7 of 36 showed nuclear staining. Asimilar staining pattern was reported in the tumor tissues, withsome variation in both the intensity and extent of immuno-staining (16). The reasons for these differences in the cytoplas-mic staining pattern are unclear. Both the antibodies used in

Fig. 2 ATM and p53 immunostaining in normal breast tissue andbreast carcinoma.Left panels,normal breast tissue stained with H&E (a;magnification,3200) and showing positive nuclear ATM staining in theinner epithelial cells of the breast ducts and negative ATM immuno-staining in the outer myoepithelial cells, using the ATM1 (b) and theATM2 antibodies (c; magnification,3400). No p53 immunoreactivitywas observed in the normal breast ducts (d; magnification,3400).Rightpanel, IDC stained with H&E (e; magnification,3200) and showinglow ATM staining using the ATM1 (f) and the ATM2 (g) antibodies,and positive p53 staining (h) in the tumor area (T) compared withlymphocytes on the same slide (N; magnification,3400).




these studies were raised against recombinant proteins. TheATM2 antibody was raised against a recombinant protein cor-responding to amino acids 2581–2599, whereas ATM-4BA wasraised against a larger fragment of the ATM protein (aminoacids 2323–2740). Thus, one might have expected that bothantibodies would give a similar staining pattern. One possibleexplanation for these differences could be if the region sur-rounding the epitope recognized by ATM2 is subject to someform of posttranslational modification, which may be involvedin the translocation of the protein from the nucleus to thecytoplasm. To date, no such modification of the ATM proteinhas been reported. In both lymphoblastoid cells and primaryfibroblasts, ATM is expressed in the nucleus but also localizesto the vesicles in the cytoplasm (34), where it interacts withb-adaptin (35).b-Adaptin is one of the components of the AP-2adaptor complex, which is involved in clathrin-mediated endo-cytosis of receptors. This interaction between ATM andb-adap-tin may play a role in the regulating mechanisms of vesiclesand/or protein transport. It has been recently shown, using theATM4-BA antibody, that a portion of ATM co-localizes withthe peroxisomal matrix protein catalase (36).

There are many potential molecular causes for a reduction

in ATM expression. Several regions on chromosome 11 havebeen identified, which are frequently lost in breast tumors (37–42). In this study, allelic imbalance on chromosome 11 wasfound in five of seven tumors and occurred within theATMgenein three cases. Interestingly, low levels of ATM expression werefound in two of these three cases; thus, the reduction could beexplained by loss of anATM allele. However, because nomutations were detected in these samples, it would seem thateither bothATMalleles were wild-type or the mutated allele waslost in the tumor. Relatively few studies have characterized themutational status of theATM gene in breast tumors. Chenet al.(12) examined the tumor tissue from four AT heterozygotesidentified in their study population of 188 patients with a familyhistory of breast cancer. In three of these tumors, both themutated and wild-type alleles were retained, and in one, themutated allele was lost, suggesting that theATM gene was notinvolved in the pathogenesis of these familial breast cancers. Incontrast, Bayet al. (13) reported the loss of the wild-typeATMallele in the breast tumor tissue of an AT heterozygote identifiedin a family with a high cancer incidence. This observation wouldsupport the hypothesis that haplo insufficiency atATM maypromote tumorigenesis and a more classic role forATM astumor-suppressor gene. A role for theATMgene in breast cancerdevelopment is also supported by the recent results of Izattet al.(43) who found that in five breast tumors from early-onsetbreast cancer patients with germline rareATM missense se-quence variants, the wild-type allele was lost.

The ATM expression profile in breast tumors resemblesthat previously reported for BRCA1. Several studies haveshown thatBRCA1gene expression at the mRNA level (44–46)and at the protein level (47–48) is significantly reduced in somesporadic breast tumors. Interestingly, low levels of BRCA1expression were also detected in sporadic breast tumors irre-spective of whether LOH in theBRCA1gene was found (49).Thus, it appears that the expression of BRCA1, and by analogyATM, can be regulated by different mechanisms, includingallelic loss and modulation of the protein expression at thetranscriptional and translational levels in sporadic breast carci-nomas. In the case of BRCA1, abnormal methylation of theBRCA1gene promoter region and preferential allelic expressionhave been reported in sporadic breast tumor samples (46, 50,51). It remains to be determined whether altered methylationpatterns of theATM promoter can explain the reduced ATMexpression seen in breast tumors. Luoet al. (52) have shownthat in lymphocytes expressing ATM, the promoter region iscompletely demethylated. However, they were unable to corre-late the methylation status and the variable ATM protein ex-pression observed in the T-PLL tumor samples in which noATM mutations were found.

The second expression profile that was examined in thesebreast tumors was that of theTP53 gene. The frequency oftumors with p53 overexpression (59% with the CM1 antibody)and that withTP53mutations (47%) are at the higher end of theranges reported where large cohorts of randomly selected breastcancer cases have been examined (18). AlthoughTP53 muta-tions have been detected at the earliest clinical stages of neo-plastic transformation, the fraction of tumors with an alteredTP53 gene is typically higher in late-stage tumors. In breasttumor tissue, a significant correlation between the presence of

Fig. 3 Variable ATM protein expression in breast carcinomas.Leftpanels,3200 magnification;Right panels,3400 magnification. ATMimmunostaining using the ATM1 or the ATM2 antibodies is absent (aandb), low (candd), moderate (eandf), or high (gandh) in a variablepercentage of epithelial cancer cells compared with lymphocytes presentin the same breast tissue section.




TP53mutations and a high histological grade (P , 0.001) hasbeen reported (53). This may explain the high prevalence ofTP53 mutations found in this study because 7 of 16 of thesamples analyzed were grade III IDCs, five of which had amutatedTP53gene.

The vast majority of theTP53mutations detected in thisstudy are among those frequently found and are in accord-ance with the publishedTP53 mutation spectra in breastcancer. They were all clustered in the core domain of p53,which contains the sequence-specific DNA-binding activityof the p53 protein (residues 102–292). This binding activityis critical for p53’s role as a transcriptional activator. Argi-nine residues in this core domain play a particularly impor-tant role in protein-DNA contacts (Arg248 and Arg273) or instabilizing the structure of the DNA-binding surface of p53(Arg175, Arg249, and Arg 282; Ref. 54). The onlyTP53mutation found in this series of samples, which did notinvolve an arginine residue, GGG3GAG at codon 279 inexon 8, which will result in a glycine3glutamic acid aminoacid change, has not been reported in the IARCTP53muta-tion database in breast tumors.

By examining both ATM and p53 profiles in the sametumor sections, this study has revealed for the first time aninteresting relationship between the expression of these twomultifunctional proteins in normal compared with tumor tissue(Table 2). In the 17 breast tumors analyzed in this study, 10cases exhibited no detectable or low ATM protein expressionand 8 tumors contained a mutatedTP53, which was associatedin three samples with low ATM protein expression. Thus, in 15of 17 of the sporadic breast carcinomas examined, the function-ality of the intricate web of signaling pathways activated inresponse to DNA damage is likely to be compromised. In thosetumors expressing a low level of ATM protein, it might beexpected that the activation of p53 through its phosphorylationby ATM’s kinase activity in response to certain forms of DNAdamage or stresses will be suboptimal. Indeed, one of thehallmark features of AT homozygote cells is reduced and de-layed p53 induction after exposure to ionizing radiation (55, 56).In lymphoblastoid cell lines from AT heterozygotes, suboptimalinduction of p53 has also been found (13, 57).8 A low level ofATM expression may also influence the modification of otherphysiological targets that are phosphorylated by the ATM ki-nase. In response to DNA double-strand breaks, the activatedATM kinase phosphorylates and activates the checkpoint kinaseChk2 (20). These activated kinases phosphorylate p53 on

serines 15 and 20, respectively, which results in its stabilizationand activation of its transcriptional activity. Khosraviet al. (58)have demonstrated that in response to DNA strand breaks, ATMalso phosphorylates Mdm2, which itself negatively regulatesp53’s stability and activity and whose gene is activated by p53.Mdm2 binds to the amino-terminus of p53, represses its trans-activation activity, and targets it to proteasome-mediated deg-radation (59–61). Thus, ATM may promote p53 activity andstability by mediating simultaneous phosphorylation of bothpartners of the p53-Mdm2 autoregulatory feedback loop. Afurther target, clearly implicated not only in the etiology ofbreast cancer but also in transcription as a transcription factorand in DNA repair through its association with Rad51/BRCA2,is the BRCA1 protein (see Ref. 62 for a recent review). ATM isrequired for the phosphorylation of BRCA1 in response toionizing radiation (63). Several other putative substrates havebeen identified, including p95/nibrin, which is altered in Nijme-gen Breakage Syndrome patients, and its protein partner Mre11,suggesting that ATM may also regulate the function of thep95-Mre11-Rad50 repair complex in response to DNA damage(64). The presence of a mutated form of the p53 protein will alsoresult in failure to activate the cellular responses normallytriggered through p53 acting as a transcription factor. Thispossible inability of breast tumor cells to activate these varioussignaling pathways because of alterations in the levels of thesetwo multifunctional proteins could be an important event in thedevelopment of sporadic breast tumors and their progression byincreasing genomic instability and must now be examined in alarger study.

ACKNOWLEDGMENTSWe are grateful to Dr Y. Shiloh for his generous gift of ATM

antibodies and Dr S. Becker-Catania for allowing us access to tissuesamples from AT patients. Helpful discussions with Dr P. Hainaut andDr B. Sylla, the expert technical assistance of M. Laval and N. Lyandrat,and the photographic assistance of G. Mollon are all gratefully acknowl-edged.

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Table 2 Classification of 17 tumors by factors affecting ATM- andp53-dependent signaling pathways

TP53mutated 8ATM expression absent or low 10Tumors with compromised ATM

or p53 signaling pathways15/17 tumorsa

a Three tumors had both aTP53mutated and a low ATM expres-sion.




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2000;6:3536-3544. Clin Cancer Res Sandra Angèle, Isabelle Treilleux, Philippe Tanière, et al. Breast Carcinomas

Genes in SporadicTP53 and ATMAbnormal Expression of the

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