Proc. Natl. Acad. Sci. USAVol. 87, pp. 7555-7559, October 1990Medical Sciences

p53 mutations in colorectal cancer(immunohistokogy/polymerase chain reaction/chemical mismatch cleavage/adenocarcinoma)

NANDA R. RODRIGUES*t, ANDREW ROWAN*, MARK E. F. SMITH*t, IAN B. KERR*, WALTER F. BODMER*,JULIAN V. GANNON§, AND DAVID P. LANE§*Director's Laboratory, Imperial Cancer Research Fund, Lincolns Inn Fields, P.O. Box 123, London WC2A 3PX, United Kingdom; §MolecularImmunochemistry Laboratory, Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Herts EN6 3LD, United Kingdom

Contributed by Walter F. Bodmer, July 9, 1990

ABSTRACT Immunohioical ining of primary colo-rectal carcinomas with antibodies specific to p53 demonstratedgross overexpression of the protein in =50% of the malignanttumors examined. Benign adenomas were all negative for p53overexpression. To determine the molular basis for this over-expression we exmine p53 protein expression in 10 coVorectalcancer cell lines. Six of the cell lines expressed high levels of p53in ELISA, cell-staining, and inmu pitation studies. Di-rect sequencing and chemical-mismatch-cleavage analysis ofp53cDNA by using the polymerase chain raction in these cell linesshowed that all cell lines that expressed high levels of p53 weresynthesizing mRNAs that encoded mutant p53 proteins. In twoof those four cell lines where p53 expression was lower, pointmutations were still detected. Thus, we conclude that overex-pression of p53 is synonymous with mutation, but some muta-tions would not be detected by a simple immunohistochemicalanalysis. Mutation of the p53 gene is one of the commonestgenetic changes in the development of human colorectal cancer.

An improved understanding of the fundamental genetics andbiology of colorectal cancer is likely to be the main basis forachieving new approaches to both its prevention and treat-ment (1). Among the most striking genetic changes are thosein the gene for p53 protein, located on chromosome 17p,initially identified because of the high frequency of allele lossin this region ofchromosome 17 (2-4). Nigro et al. (5) showedthat p53-encoding gene mutations in colorectal and othercancers occurred in specific conserved regions of the geneand might be present in well over 50o of colorectal cancers.Subsequently in our laboratories and elsewhere p53 genemutations have been found in a high proportion of lung,breast, and other tumors (6, 7).

Crawford et al. (8) showed that many human tumor-derivedcell lines express significantly elevated levels ofthe p53 productwhich can now be explained by mutated forms of p53 thatstabilize the p53 protein (9). After the demonstration that thechromosome 17 changes in colorectal cancer might be from p53mutations and account for elevated levels of the p53 product,we did initial studies with monoclonal antibodies (mAbs) to p53protein, which indicated that a high proportion of colorectalcarcinomas stained with these antibodies in histological sec-tions, whereas normal colonic epithelium was uniformly nega-tive. This result parallels similar results obtained by others (7,10, 11). In this paper we extend these observations to thedetailed analysis of p53 protein expression in 10 colorectalcancer-derived cell lines and correlate this analysis with muta-tions found in the p53 gene by direct sequencing and chemical-mismatch-cleavage analysis of cDNAs.

METHODSmAbs. PAb419 is a mAb to the simian virus 40 large tumor

antigen (12). PAb421 recognizes a denaturation-resistant

epitope located between amino acids 370 and 378 of p53protein (12, 13). PAb240 recognizes a denaturation-resistantepitope located between amino acids 156 and 335 (14, 15).PAb1801 is a mAb to human p53 protein that recognizes adenaturation-resistant epitope between amino acids 32 and 79(16). PAb1620 is a mAb to mouse and human p53 protein (17,18), which recognizes a conformationally sensitive epitopenot yet mapped.Immunochendical Studies with Anti-p53 mAbs. The ELISA

is based on a sensitive 83-galactosidase/mnouse anti-p3-galactosidase system incorporating a fluorogenic substrate(19) and using 50,000 cells in each well of an Immulon(Dynatech) 96-well plate. Cryostat sections were examinedby using a peroxidase-antiperoxidase technique (20). For p53staining, 10,000 cells were applied to poly(L-lysine) slides at500 rpm for 5 min by using a Cytospin (Shandon). Cells werefixed at 40C in chloroform/methanol, 1:1. Immunocyto-chemical staining was done as in the ELISA. Immunoprecip-itation (15) and immunoblotting of the cells were performedas described by Harlow and Lane (21).

Polymerase Chain Reaction (PCR) Sequencing and Chemi-cal-Mismatch-Cleavage Analysis. Total cellular RNA wasprepared from tissue culture cells as described in Ausubel etal. (22). One-hundred nanograms of primers E or G wasmixed with =10 ,ug of RNA (for primer sequence, see thelegend for Fig. 3). cDNA synthesis (23) and asymmetric PCR(24) were carried out as has been described. One-twentieth ofthe cDNA product was amplified by PCR between primers Aand E (regions 2 and 3) or primers D and G (regions 4 and 5).Sequencing was performed by the dideoxy chain-terminationmethod by using primers B, C, D, and F and a Sequenase 2.0kit (United States Biochemical). For chemical-mismatch-cleavage analysis, the DNA was amplified by primers D andG and purified by using Geneclean (Bio 101). One-hundrednanograms of amplified DNA from the lymphoblastoid cellline MANN was end-labeled with 60 ILCi (1 Ci = 37 GBq) of[y32-P]ATP and 10 units of polynucleotide kinase (Biolabs,Northbrook, IL). Five nanograms of the labeled probe wasmixed with 50-100 ng of the PCR-amplified DNA from thecolorectal cell lines in 0.3 M NaCl/3.5 mM MgCl2/3 mMTris HCl, pH 7.7, in a 21-.lI reaction and heated to 100'C for5 min and hybridized at 420C for 2 hr. Six microliters ofheteroduplex DNA was treated with 20 .ul offreshly prepared2.5 M hydroxylamine solution, pH 6, for 2 hr at 370C, asdescribed by Cotton et al. (25). Nine microliters of hetero-duplex DNA was treated with osmium tetroxide solution ina 25-pul reaction containing 5 mM Tris HC1, pH 8/0.5 mMEDTA/3% (vol/vol) pyridine/0.025% osmium tetroxide for 2hr at 370C (25, 26). After modification, the heteroduplexeswere ethanol-precipitated with 20 gg of the mussel glycogen,

Abbreviations: PCR, polymerase chain reaction; APC, adenomatouspolyposis coli; mAb, monoclonal antibody.tTo whom reprint requests should be addressed.lPresent address: Department of Pathology, University of MichiganMedical School, Ann Arbor, MI 48109-0602.

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7556 Medical Sciences: Rodrigues et al.

resuspended in 50 gl of fresh 1 M solution of piperidine bymixing for 30-60 sec, and incubated at 90'C for 30 min. TheDNA was then ethanol-precipitated and resuspended in 8 julof TE (10 mM Tris HCI, pH 7.7/1 mM EDTA). Two micro-liters of formamide dyes was added to the samples beforeheating to 100'C for 4 min. The samples were electrophoresedon 4-6% denaturing urea gels.

RESULTSImmunohistochemistry of p53 in Colorectal Cancer. Im-

munocytochemical reactivity of 26 sporadic colorectal car-cinomas and two from adenomatous polyposis coli (APC)patients was studied with mAbs PAb240 and PAb421. Thir-

teen of these were positive, (Fig. 1 Upper) of which threewere only focally positive. The distribution of staining waspredominantly nuclear, although some cells showed traces ofcytoplasmic reactivity. Reactivity of PAb421 was similar tothat of PAb240 but slightly weaker. Uniform negative reac-tivity was seen for all 10 cases of small tubulovillous ade-nomas, of which three were sporadic and seven were fromAPC.Immunocytochemical Staining of p53 in Colorectal Cell

Lines. Six of ten colorectal cancer cell lines stained stronglywith two different anti-p53 antibodies when an indirect immu-noperoxidase technique was used (Fig. 1 Lower, Table 1).Three more lines were weakly positive, whereas the final line

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Proc. Natl. Acad Sci. USA 87 (1990)

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Proc. Natl. Acad. Sci. USA 87 (1990) 7557

Table 1. p53 mutations in colorectal cell linesImmunoprecipitation Mutation by Mismatch

ELISA assay and immunoblotting p53 staining sequence detection,Cell line Ref. PAb240 PAb421 PAb1801 PAb240 PAb1620 PAb421 PAb240 PAb1801 analysis p53 mutation no.

WiDr 27 1344 318 2451 + +++ ++++ +++ +++++ G -A Arg-273 HisDLD-1 28 830 193 1589 - ++ +++ ++ ++ C-T Ser-241 --Phe 1SW620 29 1118 254 1872 ++ ++ ++++ +++ ++++ G-A Arg-273 - HisSW480 29 1054 260 1829 - +++ ++++ ++++ ++++ G A Arg-273 -His 2SW837 29 1867 314 Over + +±+ ++++ +++ + C-T Arg-248 TrpHT-29 30 1571 263 Over ++ +++ ++++ ++++ +++++ G-A Arg-273 His 1HCA-7 31 346 82 936 - - - + + + 3LS 174T 32 588 31 1430 - - - - - NDSW1222 29 776 251 1329 - - - ++ + + 1JW 33 764 238 1643 - - - ++ + + ND NoneMOLT4 34 98 298 - - G .A* Arg-248 GlnMANN 35 271 105 - ND

Ten human colorectal cancer cell lines, an acute lymphoblastic leukemia cell line (MOLT4), and an Epstein-Barr virus-immortalizedlymphoblastoid cell line (MANN) were analyzed for p53 protein expression in ELISA, immunoprecipitation, and cell-staining assays withanti-p53 mAbs. The p53 gene in these lines was analyzed for mutations by PCR amplification of p53-encoding cDNA, followed bychemical-mismatch-cleavage and direct sequence analysis. ND, none detected; no data entry, not determined; (-), negative. Positive (+) signalswere graded for intensity (+ to ++++ +); Over (in the ELISA assay), positive value that exceeded instrument range.*MOLT4 cell line was heterozygous in PCR sequence analysis; all other sequenced lines were homozygous at the mutant position.

(LS 174T) was completely negative, as was a set of humanlymphoblastoid cell lines including MOLT4. In the positivelines the staining pattern was predominantly nuclear, al-though some cell lines also showed evidence of p53 stainingin the cytoplasm. In most cell lines staining intensity with thetwo antibodies was concordant. However, the SW837 linestained strongly with PAb240 but only weakly with PAb1801.ELISA Analysis. p53 expression in the 10 colorectal cell

lines was also examined in an ELISA assay, by using threedifferent anti-p53 mAbs. PAb421 gave a weak signal on allcell lines, but PAb240 and PAb18O1 showed a wide range ofresponses (Table 1). With PAb240, five of the cell lines gavea particularly high reading, four showed an intermediatelevel, and one showed a low level. Broadly similar resultswere obtained with PAb1801, although less variation instrength of reaction occurred from line to line. The five celllines that reacted most strongly in the ELISA assay were allfound to react strongly in the immunocytochemical analysis.Thus, these two methods of analysis gave similar results. TheELISA assay gave background levels of binding with a seriesof lymphoblastoid cell lines.

Immunoprecipitation and Immunoblotting of p53. The 10colorectal cell lines were examined for expression of p53protein by immunoprecipitation with three different mAbs top53-PAb421, PAb1620, and PAb240. The level of p53 ineach immunoprecipitate was measured by immunoblottingwith a polyclonal anti-p53 antibody. In the 10 colorectal celllines, 6 cell lines expressed high levels of p53 (Fig. 2), and allof these lines contained a mutant p53 gene (Table 1). Theremaining 4 cell lines did not express detectable levels of thep53 protein in this assay; all of these cell lines did, however,contain p53 mRNA, as judged by the PCR reaction. Immu-noprecipitation analysis of mutant mouse p53 protein and aset ofhuman breast cancer cell lines showed that the PAb240epitope is only present on mutant p53 protein (15, 36). WhenPAb240 epitope is present, the epitope recognized byPAb1620 is lost, whereas PAb421 antibody recognizes bothforms of the protein. Different mutations affect the ratio ofPAb240-positive protein to PAbl620-positive protein (15,36). In our study four of the six mutant p53-overproducingcell lines synthesized detectable amounts ofp53 protein in thePAb240-positive conformation, whereas two cell lines did not(Fig. 2). None of the lines expressed as much p53 protein inthe PAb240 conformation as they did in the PAb1620 con-formation. This contrasts with some of the breast cell lines

examined, which showed high levels of p53 protein in thePAb240 conformation (36).

Direct Sequencing of p53 Mutation from Asymmetric PCRProducts. To determine whether the altered expression ofp53

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FIG. 2. Immunoblot of immunoprecipitates from colorectal can-cer and control simian virus 40-transformed cell line (SVK14).Extracts were immunoprecipitated with PAb419, mAb to simianvirus 40 large tumor antigen (lanes 1); PAb421, mAb to p53 that is notconformation dependent (lanes 2); PAb1620, mAb that binds to p53in normal conformation (lanes 3); PAb240, mAb that binds to p53 inmutant conformation (lanes 4). Blots were probed with rabbit anti-p53 serum.

Medical Sciences: Rodrigues et al.

7558 Medical Sciences: Rodrigues et al.

protein seen in the colorectal cell lines correlated withexpression of a mutant p53 gene, the p53 RNA from nine ofthe cell lines was sequenced. To circumvent the problem ofsequencing individual exons, we reverse-transcribed RNAfrom cells by using an antisense p53 primer and amplified thecDNA by PCR. Using asymmetric PCR, we generated single-stranded DNA with single primers. Thus, we could sequencethe most conserved region of p53-encoding gene in twosequencing runs. We also sequenced one Epstein-Barr virusimmortalized lymphoblastoid cell line (MANN) from a nor-mal individual and MOLT4, a cell line derived from anindividual with acute lymphoblastic leukemia (34). Six of thecolorectal cell lines showed point missense mutations, four ofwhich were the same (G -* A, Arg-273 -* His, Table 1). Celllines SW480 and SW620 are derived from the same patient;SW480 is derived from a colon adenocarcinoma, whereasSW620 is from a lymph node metastasis; both lines carry thesame mutation. Cell line SW837 had a point missense muta-tion (C -* T, Arg-248 Trp), and DLD-1 had a pointmissense mutation (C T, Ser-241 -* Phe). All of themutations occurred within evolutionarily conserved areas ofp53 (Fig. 3, boxes 4 and 5), and all altered amino acid residuesthat have been conserved from human to frog. All thesemutations appear homozygous because no trace of the wild-type base appears in the sequencing gel at the mutation site.MOLT4, however, contained mRNA encoding both a pointmissense mutant p53 (G -* A, Arg-248 -+ Gln, conserved box4) as well as a normal sequence at this position. The lym-phoblastoid cell line from a normal individual did not showany mutation in the conserved regions that we sequenced.

Chemical-Mismatch-Cleavage Analysis. Because direct se-quencing of PCR products can be difficult and depends onknowing the regions where mutations probably occur, wedecided to analyze the same set of cell lines by chemical-mismatch-cleavage analysis of the region amplified by prim-ers D and G. The method gave very clear results andconfirmed the sequence analysis for SW480, DLD-1, andHT-29 (Fig. 4). In addition, the method demonstrated thepresence of point mutations in the SW1222 and HCA-7 celllines, which we had not been able to identify by sequenceanalysis. SW480 had an additional band of 284 base pairs(bp), consistent with the point mutation at amino acid 309reported by Nigro et al. (5). The HT-29 cell line did not showany evidence of this second mutation (Fig. 4). The JW cellline showed no apparent mutation in the area analyzed. Therelative ease of this analysis suggests that this method is a

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FIG. 3. p53 amino acid sequence from amino acids 117-286,showing mutations (boldface letters) found by direct sequencing insix colorectal cancer cell lines. Arrows and letters show the positionand orientation of the PCR primers. Primers used were as follows: A,5'-CAGCTCCTACACCGGCGGCCCCTGCACCAG-3'; B, 5'-TCTGTCCCTTCCCAGAAAACC-3'; C, 5'-CGAAAAGTGlTITCT-GTCATCC-3'; D, 5'-TAGTGTGGTGGTGCCCTATGAGCCG-3';E, 5'-GAGCCAACCTCAGGCGGCTCATAGGGCACC-3'; F, 5'-TTGGGCAGTGCTCGCTTAGTGCTCC-3'; and G, 5'-GTGGGAG-GCTGTCAGTGGGGAACAA-3'. Residues conserved in human,mouse, rat, chicken, and Xenopus are in capital letters, and theconserved boxes (7) are underlined.

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FIG. 4. Chemical-mismatch-cleavage analysis of p53 mutation incolorectal cancer cell lines. The PCR products of six differentcolorectal cell lines were hybridized to lymphoblastoid cell lineMANN and treated with hydroxylamine and piperidine. Cleavageproducts were separated on 4% polyacrylamide sequencing gel.Fragment sizes are shown at right, and the heteroduplex is a 578-bpfragment. Lanes: 1, HCA-7; 2, SW1222; 3, JW; 4, HT-29; 5, DLD-1;6, SW480. The two products in SW480 confirm the presence of twodifferent mutations in this cell line.

sensible first choice for the genetic analysis ofp53 mutations,particularly when genomic DNA is studied. Direct sequenc-ing of the area of the mismatch can then be done if required.

DISCUSSIONThe principal finding in this study is that high-level expres-sion of p53 protein correlates with the presence of pointmissense mutations of evolutionarily conserved residues in10 colorectal cancer cell lines. The study also emphasizes thevery high frequency of p53 mutation because 8 of the 10 celllines showed clear evidence of mutation, either in the directsequence analysis or the chemical-cleavage analysis. Thisresult is consistent with recent studies on breast cancer celllines (36) and primary lung tumors (7). The implication of thisfinding is that when p53 is detected immunohistologically intumors, it is mutant. On this basis our histopathology studycan be interpreted along with the recent data ofVan Den Berget al. (10) to suggest that mutations of the p53 gene of thisspecific type are present in 50%o of overt malignant colo-rectal cancer. This figure is probably an underestimate of theproportion of such tumors carrying mutations because not allof them will be readily detected by immunohistology, asindicated here by the detection of point mutations in the p53gene in cell lines that do not express high levels ofthe protein.The data on 17p allele loss would suggest that the frequencyof p53 mutations is likely to be at least 70 or 80%. Lack ofreactivity of the antibodies both in premalignant lesions fromsporadic adenomas and polyps from APC cases indicates thatpremalignant lesions, certainly at an early stage, do not carryp53 mutations ofthe type that lead to protein overexpression.The data of Kopelovitch and Deleo (37), which suggest thatAPC patients may have elevated levels of p53 in skin fibro-blasts, could be explained by an effect of the APC gene

Proc. Natl. Acad. Sci. USA 87 (1990)

Proc. Natl. Acad. Sci. USA 87 (1990) 7559

product on control of the p53 protein level [see Bodmer et al.(38)].

All mutations that we detected in the cell lines were pointmissense mutations within two highly conserved regions ofthe p53 protein. The extreme specificity of these mutationssuggests that they have been specifically selected for, mostprobably through their effect on counteracting activity of thenormal p53 product. The fact that deletions of p53 are notselected for suggests that, at least for colorectal cancer,reducing the wild-type level ofp53 product by a factor oftwo,as would be expected in a hemizygote from gene-dosageeffects, is not enough to give a significant advantage to theoutgrowing tumor. The "dominant negative" p53 mutationsare probably selected because they sequester the normal p53gene product to an extent that results in an effective level ofavailable normal p53 product, which is much less than 50%oof normal level. In this study we have identified cell lines thatexpressed a mutant p53 gene that did not lead to grossoverexpression of the protein. This result may reflect aspecific effect of the particular mutation failing to stabilizethe protein. The high frequency of chromosome 17 allele lossindicates that, after initial selection of a p53 mutation, asignificant selective disadvantage remains associated withthe presence of the remaining wild-type p53 gene. No doubteven relatively small levels of remaining normal p53 geneproduct are enough to be disadvantageous, and that situationpresumably explains the high frequency of selection for afurther event to eliminate the remaining wild-type p53 allele.Interestingly, this is not the case for MOLT4, an acutelymphoblastic leukemia-derived cell line shown to be hetero-zygous for a mutant and a normal p53 allele. It could be thatbaseline p53 levels vary from one tissue to another and thatthis variation influences the pattern of selection for the initialmutations and for secondary loss of the remaining wild-typeallele. In some tumors, for example, the baseline level of p53may be sufficiently low that deletion of function could be thefirst step, rather than a dominant negative mutation; thissituation may occur in those tumors such as osteosarcoma(39) and chronic myeloid leukemia in blast crisis (40), inwhich p53 deletions have commonly been found.The p53 gene has now clearly been shown to be the most

commonly mutated oncogene in a wide variety of humancancers, including the most frequent adenocarcinomas. Wehope that this information will be used for earlier detection ofcancers and for new approaches to treatment, perhaps im-munologically (20) or by blocking or otherwise interferingwith mutant p53 functions.

We thank Mr. Ian Goldsmith for oligonucleotide synthesis. Ms.Cynthia Dixon for growing the colorectal cell lines, and Dr. RichardIggo for advice on PCR sequencing.

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