, . 181: 140–145 (1997)

RELATIONSHIP BETWEEN ABNORMAL p53 PROTEINAND FAILURE TO EXPRESS p21 PROTEIN IN HUMAN

BREAST CARCINOMAS

. . *, . . †Q, . ̊‡, . § . . ø*

*Genetic Department, Norwegian Radium Hospital; †Department of Pathology, Norwegian Radium Hospital; ‡Department ofSurgery, Ullevål Hospital; §Department of Surgery, Buskerud Central Hospital; and QUniversity of Oslo, Norway

SUMMARY

Alterations in the p53 protein are a common feature in most malignancies, including breast carcinomas. p53 protein alterationscontribute to malignant transformation in several ways, through genomic instability and accumulation of additional genetic alterationsin other genes, through alteration of the p53-dependent apoptotic pathway, and through downregulation of downstream effector proteinssuch as p21 (WAF1/CIP1), necessary for cell-cycle growth arrest. Cell-cycle arrest is needed to allow DNA repair after injury. Thisstudy examines the relationship between abnormalities in p53 protein and expression of p21 protein in 70 cases selected from a seriesof 212 sporadic human breast carcinomas. Immunohistochemistry (IHC) was used for detection of p53 and p21 protein expression.Constant denaturant gel electrophoresis (CDGE) was used for detection of mutations in exons 5–8 of the TP53 gene. A highly significantassociation was found between abnormalities in p53, scored as protein accumulation and/or mutations, and lack of p21 expression. p21was also shown to be downregulated in samples without p53 alterations, indicating that other mechanisms are also involved in turningoff this gene.

KEY WORDS—breast carcinoma; p53; TP53 gene; p21; immunohistochemistry; cell-cycle growth arrest

INTRODUCTION

Mammalian cells demonstrate complex responses toDNA damage, including activation of genes required forcell-cycle arrest, DNA repair, and apoptosis.1,2 Cell-cycle arrest following DNA damage is mainly mediatedby a series of negative feedback processes called check-points.3 There are at least three damage-sensitive cell-cycle checkpoints in mammalian cells: one at the G–Sboundary, one in the S-phase, and one at the G2–Mboundary.1,3–6 One of the critical components of G1arrest is the TP53 gene product.p53 protein is a 53 kD nuclear phosphoprotein with

tumour suppressor activity and it acts as a transcriptionfactor, having the ability to transactivate some genesand suppress the transcription of others. Cellular p53 isregulated by rapid protein turnover and the level presentin the nuclei of normal cells is below the sensitivity ofimmunohistochemical (IHC) detection. Mutations in theTP53 gene lead to a longer half-life of the p53 proteinand detectable p53 protein accumulation in cell nucleiis observed. Alterations in the TP53 gene have beenthought to be one of the most important factors leadingto the development of human malignancies. Mutationsin the TP53 gene have been reported in as many as10–40 per cent of breast carcinomas (for a review seeAndersen and Børresen7). Andersen et al.8 found astrong association between accumulation of p53 proteinand mutations detected by DNA analyses. A less strong

association was seen in a study performed by Lohmannet al.9p53 protein is capable of transactivating at least three

genes, MDM2,10 GADD45,11 and p21 (CIP1/WAF1).12p21 (CIP1/WAF1) is thought to be the main down-stream effector of p53 protein and is suggested tomediate p53-induced growth arrest, triggered by DNAdamage.13,14 Normally, in a cell cycle, cyclin-dependentkinases (CDKs) phosphorylate proteins such as the Rbprotein, allowing cells to enter the S-phase. In cellsexposed to DNA-damaging agents, an arrest in the G1phase of the cell cycle is seen. This arrest is thought to bemediated by p21 (WAF1/CIP1), which is increasedsecondary to an increase in p53 protein after injury toDNA. p21 (CIP1/WAF1) acts by binding to CDK–cyclin complexes, inhibiting their kinase activity andthereby leading to lack of phosphorylation of crucialproteins. This results in blockage of entry into theS-phase, allowing sufficient time for repair.15In several studies, most of them on cell lines, p53

protein accumulation in response to DNA-damagingagents has been compared with the p21 (CIP1/WAF1)response. A strong association has been found betweenDNA damage, p53 protein accumulation, and p21expression.13,15–18 p21 (CIP1/WAF1) protein expressionhas been detected in cells with wild-type TP53, but notin cells lacking p53 protein activity.The aim of the present study was to examine the

relationship between TP53 gene mutations, p53 pro-tein accumulation, and expression of p21 protein in aseries of sporadic human breast carcinomas, in orderto investigate the importance of this pathway in thepathogenesis of these tumours.

Addressee for correspondence: A. L. Børresen, Department ofGenetics, Institute of Cancer Research, Montebello, N-0310 Oslo,Norway.

CCC 0022–3417/97/020140–06 Received 12 April 1996? 1997 by John Wiley & Sons, Ltd. Accepted 25 July 1996

MATERIALS AND METHODS

Patient and tumour materialMaterial for this study was obtained from 212 pri-

mary breast carcinoma patients admitted to UllevålHospital during the period from 1987 to 1994. Mean ageat diagnosis was 64·5 years (range 28–91 years). Lymphnode dissection was performed in 188 patients. Out ofthese, 110 (58·5 per cent) patients were lymph node-negative and 78 (41·5 per cent) lymph node-positive atthe time of surgery. Twenty-five were classified as in-vasive lobular carcinomas, 138 as invasive ductal carci-nomas, and 31 as other types. Eighteen of the tumourshad no histological classification. Fifty-eight of 194tumours with histological classification (29·9 per cent)were classified as grade 3, 119 (63·3 per cent) as grade 2,and 17 (8·88 per cent) as grade 1 tumours. Grading ofthe tumours was based on the recommendations ofElston and Ellis.19 Clinical stage was based on the TNMclassification from 1988.20 All samples included inthis study were judged after histological evaluation tocontain more than 20 per cent tumour tissue. Meanobservation time was 2·8 years (range 0–4·4 years).Fifty-nine patients (24·8 per cent) had recurrences ordistant metastases (to liver, lungs, skeleton, CNS). Six-teen patients were dead of disseminated breast cancerand four of other causes.

Immunohistochemical detection of p53 protein

Fresh tumour tissue obtained at surgery was frozen inliquid nitrogen and stored at "70)C. Frozen sectionswere cut and immunostaining for p53 protein wasperformed, applying the avidin–biotin–peroxidase com-plex (ABC) methods.21 Briefly, the frozen sections wereair-dried and fixed in cold acetone at 4)C for 10 min.After preincubation with mouse serum for 20 min atroom temperature, the sections were incubated with themonoclonal antibody PAB 1801 (Oncogene Science),dilution 1–100, for 16–22 h at 4)C. The sections werethen incubated with the biotin-labelled secondary anti-body for 30 min and washed in phosphate-bufferedsaline (PBS), followed by incubation with ABC for 60min. The peroxidase reaction was carried out usingdiaminobenzidine as chromogen. All series includedpositive and negative controls. Only cells with nuclearstaining were scored as p53 protein-immunopositive.The amount of immunopositive cells was semi-quantitatively estimated: grade + corresponded to 1–10per cent, grade ++ to 10–50 per cent, and grade +++to more than 50 per cent positive cells.

Mutation analysis

DNA was extracted from frozen tumour tissue byphenol/chloroform followed by ethanol precipitation.22Amplification of exons 5–8 of the TP53 gene wasperformed by the polymerase chain reaction (PCR),using primers and conditions as described previously.23Exons 5–8 cover the evolutionarily conserved region ofthe gene in which more than 80 per cent of the reportedmutations have been found. The PCR products were

screened for TP53 mutations using constant denaturantgel electrophoresis (CDGE).23 Samples with aberrantlymigrating bands were confirmed by perpendicular dena-turing gradient gel electrophoresis (DGGE). All PCRfragments with a mobility different from normal DNAin CDGE and DGGE analyses were considered to bemutants. Mutant samples were submitted to PCR withone biotinylated primer and were sequenced partlyby manual sequencing and by an automatic sequencer(ABI 373).

Immunohistochemical detection of p21 (WAF1/CIP1)

The antibody used against p21 (WAF1, Ab-1 fromOncogene Science) did not function on frozen sections,but worked well on paraffin sections. Four to six ìmthick sections from paraffin-embedded material from 70selected cases were mounted on coated slides. The caseswere selected to include all p53 protein-positive samples(31), all samples negative for p53 by IHC but witha mutation in the TP53 gene (7), and 32 randomlysampled tumours with no detectable alterations in p53protein expression. After antigen retrieval by microwaveoven, the same procedure was followed as for p53protein immunostaining. All series included positive andnegative controls. The amount of immunoreactive cellswas semi-quantitatively estimated: grade + corre-sponded to 5–10 per cent positive cells, grade ++ to10–50 per cent positive cells, and grade +++ to morethan 50 per cent positive cells.

Statistical methods

Statistical analysis was performed by the chi-squaretest. The level of statistical significance was P<0·05.

RESULTS

p53 protein accumulation and TP53 gene mutationanalysisThirty-one (14·6 per cent) of the 212 samples were

judged to have accumulation of p53 protein by IHC, ofwhich six were +, ten ++, and 15 +++. Twenty-five ofthese were found in invasive ductal carcinomas, twoin invasive lobular carcinomas, and four in tumourswhere histological diagnosis was not available.Mutation analyses of exons 5–8 using CDGE/DGGE

revealed mutations in 25 (11·8 per cent) of the samples.In 21 of these samples, sequence analysis revealed theexact nature of the mutation (Table I). An example ofthe results of CDGE/DGGE analysis is shown in Figs1A and 1B. In four samples, the sequence change couldnot be determined, due to the small amount of mutatedcells in the tissue samples. We found 15 mutations ininvasive ductal carcinomas, four in other types, and sixin tumours without histological diagnosis. In the inva-sive lobular carcinomas, no mutations were found. In 13samples with p53 protein accumulation a mutationcould not be found (Tables I and II). When comparingp53 protein accumulation with mutation in the TP53gene, a significant association was found (P<0·001,

141ABNORMAL p53 PROTEIN AND REDUCED EXPRESSION OF p21 PROTEIN

Table II). When comparing p53 protein accumulationwith the tumour grade, we found one example in grade 1tumours, 15 in grade 2, and 15 in grade 3 (Table III).TP53 gene mutation in relation to grade gave oneexample in grade 1 tumours, 15 in grade 2, and nine ingrade 3 (Table III). The fraction of tumours with p53alteration scored as accumulation or mutation was thesame in grade 1 and grade 2 tumours. In grade 3tumours, however, there was a greater fraction of p53protein accumulation than TP53 gene mutations.Tumours with mutations not detected by IHC weredistributed equally in grade 2 and grade 3 tumours.Tumours with p53 alteration not detected by DNAanalysis, but detected by IHC, were clustered mostly ingrade 3 (Table III).

p21 protein expression analysis

The expression of the p21 (WAF1/CIP1) protein wasanalysed in 70 selected tumour samples. No p21 proteinexpression was detected in 28 tumours, low expression(+) was detected in 23, and high expression (++/+++)was detected in 19. When comparing p21 expressionwith p53 accumulation (Table IV), a significant associ-ation was found between high p53 protein accumulationand downregulation of p21 expression (P<0·001). Whencomparing p21 expression with gene mutation in theTP53 gene, a similar association was seen (Table V).When combining p53 accumulation and TP53 genemutation, 37 of the 38 samples (97·4 per cent) with p53alteration had downregulation of p21 (Table VI).Among the 31 tumours with undetectable p53 alteration,14 (40·6 per cent) had downregulation of the p21 protein.An example of p53 and p21 protein immunostaining isshown in Figs 2A and 2B.

DISCUSSION

In this study, a strong association was found betweenalteration of p53 protein and p21 protein (CIP1/WAF1)expression. This observation correlates well with pre-vious studies in experimental systems.15,18 Gudas et al.24have recently demonstrated on breast cancer cell linesthat after DNA damage, the p21 level correlates wellwith the amount of normal p53 protein. As shownabove, tumours with a high degree of accumulation ofp53 protein showed the weakest staining for p21 protein.When comparing p21 protein expression with mutationsof the TP53 gene alone, the association was not particu-larly strong, suggesting that mutations outside thescreened area, or mechanisms other than mutation ofthe TP53 gene, may have led to a dysfunctional p53protein, with respect to its ability to induce p21 expres-sion. Some mutants are able to turn on the p21 protein,but a clear relationship is not seen between a specificmutant and the ability to act as a transcription factor forp21. For example, the same mutation, changing codon273 from Arg]His, was found in three differenttumours (sample Nos 164, 252, and 274), but the resultsregarding p53 protein accumulation and p21 proteinexpression in these tumours were different. In No. 164,high p53 protein accumulation and no p21 proteinexpression were seen; in No. 252, no p53 protein accu-mulation was detected by IHC and low p21 proteinexpression was seen; in No. 274, a high degree of p53protein accumulation was detected together with lowp21 protein expression. All three tumours had variableamounts of wild-type p53 present, as judged by CDGE/DGGE analysis, but it was impossible to evaluatewhether the wild-type gene was present in the cells thatcontained the mutation, or in normal cells contaminat-ing the tumour. Another explanation of the differentresults regarding p53 protein accumulation in these threetumours with the same mutation could be that p53protein is modified by other proteins. Expression of p21protein was also different in these three tumours. Oneexplanation of this difference could be that p21 has beenmodulated by a p53-independent pathway.

Table I—Breast carcinoma with detectable TP53 gene muta-tions and/or p53 protein accumulation

SampleNo.

TP53 mutation

p53IHC

p21IHC

Basechange

Codonchange

Aminoacid

UT 009 CGT]TGT 273 Arg]Cys ++ 0U-T032 No mutation detected + +U-T038 GCC]GTC 138 Ala]Val ++ +U-T049 AAC]AGC 239 Asn]Ser ++ 0U-T053 No mutation detected +++ 0U-T056 AGG]ATG 249 Arg]Met 0 +U-T065 CGT]TGT 273 Arg]Cys +++ 0U-T080 GCC]GTC 138 Ala]Val 0 0U-T083 No mutation detected + 0U-T096 No mutation detected +++ 0U-T098 No mutation detected ++ 0U-T099 GTG]CTG 173 Val]Leu +++ 0U-T102 TGT]TAT 238 Cys]Tyr 0 0U-T106 Aberrant migrating band in exon 5 +++ 0U-T109 Aberrant migrating band in exon 7 +++ 0U-T125 No mutation detected + 0U-T129 TGT]CGT 275 Cys]Arg +++ 0U-T154 CGG]TGG 282 Arg]Trp ++ 0U-T155 No mutation detected + +U-T164 CGT]CAT 273 Arg]His +++ 0U-T167 ATC]AGC 251 Ile]Ser ++ 0U-T173 No mutation detected +++ 0U-T174 Aberrant migrating band in exon 5 +++ 0U-T177 No mutation detected ++ +U-T188 No mutation detected +++ 0U-T198 GTG]CTG 173 Val]Leu ++ +U-T200 No mutation detected ++ +U-T202 No mutation detected +++ 0U-T203 Aberrant migrating band in exon 5 + 0U-T209 CGG]TGG 282 Arg]Trp + 0U-T212 TAC]GAC 126 Tyr]Asp +++ 0U-T215 CCT]CTT 278 Pro]Leu 0 +U-T221 AAC]AGC 239 Asn]Ser +++ 0U-T238 GGC]GAC 244 Gly]Asp 0 0U-T250 CGG]CAG 248 Arg]Gln 0 0U-T252 CGT]CAT 273 Arg]His 0 +U-T254 No mutation detected +++ ++U-T274 CGT]CAT 273 Arg]His ++ +

IHC=immunohistochemistry.

142 I. K. BUKHOLM ET AL.

The fraction of abnormal p53 protein increased withincreasing tumour grade. This was the case for bothTP53 gene mutations and p53 protein accumulation.However, in grade 3 tumours, there was a difference inthe frequency of TP53 gene mutations compared withp53 protein accumulation, although not statisticallysignificant. These results indicate that mechanisms otherthan mutation of the TP53 gene are more significant forinactivation of p53 protein in grade 3 tumours. Modifi-cation of p53 protein by binding to other oncogene

products, such as MDM2 as suggested by Fan et al.,may be one such mechanism.16The overall incidence of detection of abnormal p53

protein is relatively low in this study. The incidence hasvaried in different studies. The reason for the lowincidence may be that most of the tumours in this studyare of grade 1 and 2 (>70 per cent), indicating an earlyand more benign stage of the cancers.

Fig. 1—(A) Constant denaturant gel electrophoresis (CDGE) of PCR-amplified exon 8 fragments. Lane a: tumour No. 274; lane b:mutant control with codon 273 (CGT]CAT, Arg]His). The rest of the lanes show tumours with wild-type sequence. Runningconditions: 12·5 per cent polyacrylamide gel containing 47 per cent denaturant; running time 2·5 h at 56)C at 80 V constant.(B) DGGE of PCR-amplified exon 8 fragment of tumour sample No. 274 analysed on 12·5 per cent polyacrylamide gel with a 10–80per cent gradient of denaturant. Running time 2 h at 56)C with 80 V constant. 1: heteroduplex bands; 2: homoduplex mutant band;3: homoduplex wild-type band

Table II—Relationship between p53 protein accumulation andTP53 gene mutation in 212 breast carcinomas

p53 protein accumulation0 + ++ +++

TP53 gene mutation 0 173 4 3 6+ 7 2 7 9

Comparison between p53 protein accumulation scored as 0 or + to+++ vs. TP53 mutation gave P<0·001.

Table III—Relationship between tumour grade, TP53 genemutation analysis, and p53 protein accumulation

GradeTP53 genemutation

p53 proteinaccumulation P

1 1/17 (5·8%) 1/17 (5·8%) NS2 15/119 (12·6%) 15/119 (12·6%) NS3 9/58 (15·5%) 15/58 (25·9%) (P>0·05)

NS=not significant.

Table IV—Relationship between p53 protein accumulationand expression of p21 protein

p21 protein expression0 + ++ +++

p53 accumulation 0 4 18 14 4+ 4 2 0 0++ 5 5 0 0+++ 14 0 1 0

Comparison between p53 protein scored as 0 or + to +++ vs. p21expression scored as 0 or + to +++ gave P<0·001.

Table V—Relationship between TP53 gene mutation and p21protein expression

p21 protein IHC positive0 + ++ +++

TP53 gene normal 11 17 13 3TP53 gene mutation 19 6 0 0

Comparison between TP53 gene mutation vs. p21 protein expres-sion scored as 0 or + to +++ gave P<0·001.

143ABNORMAL p53 PROTEIN AND REDUCED EXPRESSION OF p21 PROTEIN

Data from the present study suggest that inactivationof p53 protein is strongly associated with tumour aggres-siveness. We found an increasing rate of abnormal p53protein with increasing grade of the tumour. Theabsence of p21 protein and the resulting altered G1arrest and insufficient DNA repair probably constituteone contributing factor.The results from this study indicate that IHC is a

relatively reliable method for testing the functionalproperties of p53 protein, such as its ability to inducep21 protein. DNA analysis, however, gives additionalinformation. Some mutations of the TP53 gene lead tono protein and therefore normal IHC staining. In othercircumstances, modifications of p53 protein are ofimportance, but these modifications are not detected byDNA analysis. We therefore believe that in futurestudies, both methods should be used to give optimalinformation.

In summary, data from the present study demonstratethat in breast carcinoma, p21 protein expression corre-lates well with normal p53 protein function. Furtherstudies are required to support these findings and toevaluate their relevance to clinical behaviour.

ACKNOWLEDGEMENTS

This study was supported by the NorwegianCancer Society. The excellent technical assistance of MsEldri U. Due and Ms Hilde Johnsen is gratefullyacknowledged.

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