To determine the molular basis for this over- expression we exmine p53 protein expression in 10 coVorectal cancer cell lines. Six of the cell linesexpressed high ...
Proc. Natl. Acad. Sci. USA Vol. 87, pp. 7555-7559, October 1990
Medical 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; §Molecular Immunochemistry 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
Immunohioical ining of primary coloABSTRACT rectal carcinomas with antibodies specific to p53 demonstrated gross overexpression of the protein in =50% of the malignant tumors examined. Benign adenomas were all negative for p53 overexpression. To determine the molular basis for this overexpression we exmine p53 protein expression in 10 coVorectal cancer cell lines. Six of the cell lines expressed high levels of p53 in ELISA, cell-staining, and inmu pitation studies. Direct sequencing and chemical-mismatch-cleavage analysis ofp53 cDNA by using the polymerase chain raction in these cell lines showed that all cell lines that expressed high levels of p53 were synthesizing mRNAs that encoded mutant p53 proteins. In two of those four cell lines where p53 expression was lower, point mutations were still detected. Thus, we conclude that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis. Mutation of the p53 gene is one of the commonest genetic changes in the development of human colorectal cancer.
epitope located between amino acids 370 and 378 of p53 protein (12, 13). PAb240 recognizes a denaturation-resistant epitope located between amino acids 156 and 335 (14, 15). PAb1801 is a mAb to human p53 protein that recognizes a denaturation-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 epitope not yet mapped. Immunochendical Studies with Anti-p53 mAbs. The ELISA is based on a sensitive 83-galactosidase/mnouse anti-p3galactosidase system incorporating a fluorogenic substrate (19) and using 50,000 cells in each well of an Immulon (Dynatech) 96-well plate. Cryostat sections were examined by using a peroxidase-antiperoxidase technique (20). For p53 staining, 10,000 cells were applied to poly(L-lysine) slides at 500 rpm for 5 min by using a Cytospin (Shandon). Cells were fixed at 40C in chloroform/methanol, 1:1. Immunocytochemical staining was done as in the ELISA. Immunoprecipitation (15) and immunoblotting of the cells were performed as described by Harlow and Lane (21). Polymerase Chain Reaction (PCR) Sequencing and Chemical-Mismatch-Cleavage Analysis. Total cellular RNA was prepared from tissue culture cells as described in Ausubel et al. (22). One-hundred nanograms of primers E or G was mixed with =10 ,ug of RNA (for primer sequence, see the legend for Fig. 3). cDNA synthesis (23) and asymmetric PCR (24) were carried out as has been described. One-twentieth of the cDNA product was amplified by PCR between primers A and E (regions 2 and 3) or primers D and G (regions 4 and 5). Sequencing was performed by the dideoxy chain-termination method by using primers B, C, D, and F and a Sequenase 2.0 kit (United States Biochemical). For chemical-mismatchcleavage analysis, the DNA was amplified by primers D and G and purified by using Geneclean (Bio 101). One-hundred nanograms of amplified DNA from the lymphoblastoid cell line 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 was mixed with 50-100 ng of the PCR-amplified DNA from the colorectal cell lines in 0.3 M NaCl/3.5 mM MgCl2/3 mM Tris HCl, pH 7.7, in a 21-.lI reaction and heated to 100'C for 5 min and hybridized at 420C for 2 hr. Six microliters of heteroduplex DNA was treated with 20 .ul of freshly prepared 2.5 M hydroxylamine solution, pH 6, for 2 hr at 370C, as described by Cotton et al. (25). Nine microliters of heteroduplex DNA was treated with osmium tetroxide solution in a 25-pul reaction containing 5 mM Tris HC1, pH 8/0.5 mM EDTA/3% (vol/vol) pyridine/0.025% osmium tetroxide for 2 hr at 370C (25, 26). After modification, the heteroduplexes were ethanol-precipitated with 20 gg of the mussel glycogen,
An improved understanding of the fundamental genetics and biology of colorectal cancer is likely to be the main basis for achieving new approaches to both its prevention and treatment (1). Among the most striking genetic changes are those in the gene for p53 protein, located on chromosome 17p, initially identified because of the high frequency of allele loss in this region of chromosome 17 (2-4). Nigro et al. (5) showed that p53-encoding gene mutations in colorectal and other cancers occurred in specific conserved regions of the gene and might be present in well over 50o of colorectal cancers. Subsequently in our laboratories and elsewhere p53 gene mutations have been found in a high proportion of lung, breast, and other tumors (6, 7). Crawford et al. (8) showed that many human tumor-derived cell lines express significantly elevated levels of the p53 product which can now be explained by mutated forms of p53 that stabilize the p53 protein (9). After the demonstration that the chromosome 17 changes in colorectal cancer might be from p53 mutations and account for elevated levels of the p53 product, we did initial studies with monoclonal antibodies (mAbs) to p53 protein, which indicated that a high proportion of colorectal carcinomas stained with these antibodies in histological sections, whereas normal colonic epithelium was uniformly negative. This result parallels similar results obtained by others (7, 10, 11). In this paper we extend these observations to the detailed analysis of p53 protein expression in 10 colorectal cancer-derived cell lines and correlate this analysis with mutations found in the p53 gene by direct sequencing and chemicalmismatch-cleavage analysis of cDNAs.
METHODS mAbs. PAb419 is a mAb to the simian virus 40 large tumor antigen (12). PAb421 recognizes a denaturation-resistant
Abbreviations: PCR, polymerase chain reaction; APC, adenomatous polyposis coli; mAb, monoclonal antibody. tTo whom reprint requests should be addressed. lPresent address: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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resuspended in 50 gl of fresh 1 M solution of piperidine by mixing for 30-60 sec, and incubated at 90'C for 30 min. The DNA was then ethanol-precipitated and resuspended in 8 jul of TE (10 mM Tris HCI, pH 7.7/1 mM EDTA). Two microliters of formamide dyes was added to the samples before heating to 100'C for 4 min. The samples were electrophoresed on 4-6% denaturing urea gels.
teen of these were positive, (Fig. 1 Upper) of which three were only focally positive. The distribution of staining was predominantly nuclear, although some cells showed traces of cytoplasmic reactivity. Reactivity of PAb421 was similar to that of PAb240 but slightly weaker. Uniform negative reactivity was seen for all 10 cases of small tubulovillous adenomas, of which three were sporadic and seven were from
RESULTS Immunohistochemistry of p53 in Colorectal Cancer. Immunocytochemical reactivity of 26 sporadic colorectal carcinomas and two from adenomatous polyposis coli (APC) patients was studied with mAbs PAb240 and PAb421. Thir-
APC. Immunocytochemical Staining of p53 in Colorectal Cell Lines. Six of ten colorectal cancer cell lines stained strongly with two different anti-p53 antibodies when an indirect immunoperoxidase technique was used (Fig. 1 Lower, Table 1). Three more lines were weakly positive, whereas the final line A&M
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FIG. 1. (Upper) Immunohistochemical detection of p53 protein in a cryostat section of adenocarcinoma with anti-p53 mAb PAb240 and peroxidase-antiperoxidase detection. Intense nuclear staining is evident in the tumor but absent from the surrounding stroma. (Lower) Immunocytochemical detection of p53 protein in the nucleus of a Cytospin preparation of HT-29 colorectal cancer cell line. The staining method is the same as for Upper.
Medical Sciences: Rodrigues
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Proc. Natl. Acad. Sci. USA 87 (1990)
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Table 1. p53 mutations in colorectal cell lines Immunoprecipitation Mismatch Mutation by p53 staining and immunoblotting ELISA assay detection, sequence no. p53 mutation analysis Cell line Ref. PAb240 PAb421 PAb1801 PAb240 PAb1620 PAb421 PAb240 PAb1801 His Arg-273 +++++ G +++ -A ++++ + +++ 2451 318 27 1344 WiDr 1 Ser-241 --Phe C -T ++ +++ ++ ++ 1589 193 830 28 DLD-1 Arg-273 - His G -A ++++ ++ ++ ++++ +++ 254 1872 29 1118 SW620 2 +++ ++++ ++++ ++++ G A 260 1829 1054 29 SW480 Arg-273 -His + C -T + +±+ ++++ +++ 314 Over 1867 29 SW837 Arg-248 Trp 1 ++++ ++++ +++++ Arg-273 His ++ +++ G -A Over 1571 263 HT-29 30 3 + ++ 346 82 936 31 HCA-7 ND 1430 588 31 LS 174T 32 1 ++ ++ 1329 251 776 SW1222 29 None ++ ++ ND 1643 764 238 33 JW G .A* Arg-248 Gln 98 298 MOLT4 34 ND 271 105 MANN 35 Ten human colorectal cancer cell lines, an acute lymphoblastic leukemia cell line (MOLT4), and an Epstein-Barr virus-immortalized lymphoblastoid cell line (MANN) were analyzed for p53 protein expression in ELISA, immunoprecipitation, and cell-staining assays with anti-p53 mAbs. The p53 gene in these lines was analyzed for mutations by PCR amplification of p53-encoding cDNA, followed by chemical-mismatch-cleavage and direct sequence analysis. ND, none detected; no data entry, not determined; (-), negative. Positive (+) signals were 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 human lymphoblastoid cell lines including MOLT4. In the positive lines the staining pattern was predominantly nuclear, although some cell lines also showed evidence of p53 staining in the cytoplasm. In most cell lines staining intensity with the two antibodies was concordant. However, the SW837 line stained 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 three different anti-p53 mAbs. PAb421 gave a weak signal on all cell lines, but PAb240 and PAb18O1 showed a wide range of responses (Table 1). With PAb240, five of the cell lines gave a particularly high reading, four showed an intermediate level, and one showed a low level. Broadly similar results were obtained with PAb1801, although less variation in strength of reaction occurred from line to line. The five cell lines that reacted most strongly in the ELISA assay were all found to react strongly in the immunocytochemical analysis. Thus, these two methods of analysis gave similar results. The ELISA assay gave background levels of binding with a series of lymphoblastoid cell lines. Immunoprecipitation and Immunoblotting of p53. The 10 colorectal cell lines were examined for expression of p53 protein by immunoprecipitation with three different mAbs to p53-PAb421, PAb1620, and PAb240. The level of p53 in each immunoprecipitate was measured by immunoblotting with a polyclonal anti-p53 antibody. In the 10 colorectal cell lines, 6 cell lines expressed high levels of p53 (Fig. 2), and all of these lines contained a mutant p53 gene (Table 1). The remaining 4 cell lines did not express detectable levels of the p53 protein in this assay; all of these cell lines did, however, contain p53 mRNA, as judged by the PCR reaction. Immunoprecipitation analysis of mutant mouse p53 protein and a set of human breast cancer cell lines showed that the PAb240 epitope is only present on mutant p53 protein (15, 36). When PAb240 epitope is present, the epitope recognized by PAb1620 is lost, whereas PAb421 antibody recognizes both forms of the protein. Different mutations affect the ratio of PAb240-positive protein to PAbl620-positive protein (15, 36). In our study four of the six mutant p53-overproducing cell lines synthesized detectable amounts of p53 protein in the PAb240-positive conformation, whereas two cell lines did not (Fig. 2). None of the lines expressed as much p53 protein in the PAb240 conformation as they did in the PAb1620 conformation. This contrasts with some of the breast cell lines
examined, which showed high levels of p53 protein in the PAb240 conformation (36). Direct Sequencing of p53 Mutation from Asymmetric PCR Products. To determine whether the altered expression of p53 SVK14
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FIG. 2. Immunoblot of immunoprecipitates from colorectal cancer and control simian virus 40-transformed cell line (SVK14). Extracts were immunoprecipitated with PAb419, mAb to simian virus 40 large tumor antigen (lanes 1); PAb421, mAb to p53 that is not
conformation dependent (lanes 2); PAb1620, mAb that binds to p53 in normal conformation (lanes 3); PAb240, mAb that binds to p53 in mutant conformation (lanes 4). Blots were probed with rabbit anti-
p53 serum.
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protein seen in the colorectal cell lines correlated with expression of a mutant p53 gene, the p53 RNA from nine of the cell lines was sequenced. To circumvent the problem of sequencing individual exons, we reverse-transcribed RNA from cells by using an antisense p53 primer and amplified the cDNA by PCR. Using asymmetric PCR, we generated singlestranded DNA with single primers. Thus, we could sequence the most conserved region of p53-encoding gene in two sequencing runs. We also sequenced one Epstein-Barr virus immortalized lymphoblastoid cell line (MANN) from a normal individual and MOLT4, a cell line derived from an individual with acute lymphoblastic leukemia (34). Six of the colorectal cell lines showed point missense mutations, four of which were the same (G -* A, Arg-273 -* His, Table 1). Cell lines SW480 and SW620 are derived from the same patient; SW480 is derived from a colon adenocarcinoma, whereas SW620 is from a lymph node metastasis; both lines carry the same mutation. Cell line SW837 had a point missense mutation (C -* T, Arg-248 Trp), and DLD-1 had a point missense mutation (C T, Ser-241 -* Phe). All of the mutations occurred within evolutionarily conserved areas of p53 (Fig. 3, boxes 4 and 5), and all altered amino acid residues that have been conserved from human to frog. All these mutations appear homozygous because no trace of the wildtype base appears in the sequencing gel at the mutation site. MOLT4, however, contained mRNA encoding both a point missense mutant p53 (G -* A, Arg-248 -+ Gln, conserved box 4) as well as a normal sequence at this position. The lymphoblastoid cell line from a normal individual did not show any mutation in the conserved regions that we sequenced. Chemical-Mismatch-Cleavage Analysis. Because direct sequencing of PCR products can be difficult and depends on knowing the regions where mutations probably occur, we decided to analyze the same set of cell lines by chemicalmismatch-cleavage analysis of the region amplified by primers D and G. The method gave very clear results and confirmed the sequence analysis for SW480, DLD-1, and HT-29 (Fig. 4). In addition, the method demonstrated the presence of point mutations in the SW1222 and HCA-7 cell lines, which we had not been able to identify by sequence analysis. SW480 had an additional band of 284 base pairs (bp), consistent with the point mutation at amino acid 309 reported by Nigro et al. (5). The HT-29 cell line did not show any evidence of this second mutation (Fig. 4). The JW cell line showed no apparent mutation in the area analyzed. The relative ease of this analysis suggests that this method is a aa117 A
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FIG. 3. p53 amino acid sequence from amino acids 117-286, showing mutations (boldface letters) found by direct sequencing in six colorectal cancer cell lines. Arrows and letters show the position and orientation of the PCR primers. Primers used were as follows: A, 5'-CAGCTCCTACACCGGCGGCCCCTGCACCAG-3'; B, 5'TCTGTCCCTTCCCAGAAAACC-3'; C, 5'-CGAAAAGTGlTITCTGTCATCC-3'; D, 5'-TAGTGTGGTGGTGCCCTATGAGCCG-3'; E, 5'-GAGCCAACCTCAGGCGGCTCATAGGGCACC-3'; F, 5'TTGGGCAGTGCTCGCTTAGTGCTCC-3'; and G, 5'-GTGGGAGGCTGTCAGTGGGGAACAA-3'. Residues conserved in human, mouse, rat, chicken, and Xenopus are in capital letters, and the conserved boxes (7) are underlined.
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FIG. 4. Chemical-mismatch-cleavage analysis of p53 mutation in colorectal cancer cell lines. The PCR products of six different colorectal cell lines were hybridized to lymphoblastoid cell line MANN and treated with hydroxylamine and piperidine. Cleavage products were separated on 4% polyacrylamide sequencing gel. Fragment sizes are shown at right, and the heteroduplex is a 578-bp fragment. 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 two different mutations in this cell line.
sensible first choice for the genetic analysis of p53 mutations, particularly when genomic DNA is studied. Direct sequencing of the area of the mismatch can then be done if required.
DISCUSSION The principal finding in this study is that high-level expression of p53 protein correlates with the presence of point missense mutations of evolutionarily conserved residues in 10 colorectal cancer cell lines. The study also emphasizes the very high frequency of p53 mutation because 8 of the 10 cell lines showed clear evidence of mutation, either in the direct sequence analysis or the chemical-cleavage analysis. This result is consistent with recent studies on breast cancer cell lines (36) and primary lung tumors (7). The implication of this finding is that when p53 is detected immunohistologically in tumors, it is mutant. On this basis our histopathology study can be interpreted along with the recent data of Van Den Berg et al. (10) to suggest that mutations of the p53 gene of this specific type are present in 50%o of overt malignant colorectal cancer. This figure is probably an underestimate of the proportion of such tumors carrying mutations because not all of them will be readily detected by immunohistology, as indicated here by the detection of point mutations in the p53 gene in cell lines that do not express high levels of the protein. The data on 17p allele loss would suggest that the frequency of p53 mutations is likely to be at least 70 or 80%. Lack of reactivity of the antibodies both in premalignant lesions from sporadic adenomas and polyps from APC cases indicates that premalignant lesions, certainly at an early stage, do not carry p53 mutations of the type that lead to protein overexpression. The data of Kopelovitch and Deleo (37), which suggest that APC patients may have elevated levels of p53 in skin fibroblasts, could be explained by an effect of the APC gene
Medical Sciences: Rodrigues
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product on control of the p53 protein level [see Bodmer et al.
(38)].
All mutations that we detected in the cell lines were point missense mutations within two highly conserved regions of the p53 protein. The extreme specificity of these mutations suggests that they have been specifically selected for, most probably through their effect on counteracting activity of the normal p53 product. The fact that deletions of p53 are not selected for suggests that, at least for colorectal cancer, reducing the wild-type level of p53 product by a factor of two, as would be expected in a hemizygote from gene-dosage effects, is not enough to give a significant advantage to the outgrowing tumor. The "dominant negative" p53 mutations are probably selected because they sequester the normal p53 gene product to an extent that results in an effective level of available normal p53 product, which is much less than 50%o of normal level. In this study we have identified cell lines that expressed a mutant p53 gene that did not lead to gross overexpression of the protein. This result may reflect a specific effect of the particular mutation failing to stabilize the protein. The high frequency of chromosome 17 allele loss indicates that, after initial selection of a p53 mutation, a significant selective disadvantage remains associated with the presence of the remaining wild-type p53 gene. No doubt even relatively small levels of remaining normal p53 gene product are enough to be disadvantageous, and that situation presumably explains the high frequency of selection for a further event to eliminate the remaining wild-type p53 allele. Interestingly, this is not the case for MOLT4, an acute lymphoblastic leukemia-derived cell line shown to be heterozygous for a mutant and a normal p53 allele. It could be that baseline p53 levels vary from one tissue to another and that this variation influences the pattern of selection for the initial mutations and for secondary loss of the remaining wild-type allele. In some tumors, for example, the baseline level of p53 may be sufficiently low that deletion of function could be the first step, rather than a dominant negative mutation; this situation may occur in those tumors such as osteosarcoma (39) and chronic myeloid leukemia in blast crisis (40), in which 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 human cancers, including the most frequent adenocarcinomas. We hope that this information will be used for earlier detection of cancers and for new approaches to treatment, perhaps immunologically (20) or by blocking or otherwise interfering with mutant p53 functions. We thank Mr. Ian Goldsmith for oligonucleotide synthesis. Ms. Cynthia Dixon for growing the colorectal cell lines, and Dr. Richard Iggo for advice on PCR sequencing. 1. Bodmer, W. F. (1988) Cancer Surv. 7, 239-250. 2. Fearon, E. R., Hamilton, S. R. & Vogelstein, B. (1987) Science 238, 193-197. 3. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., Preisinger, A. C., Leppert, M., Nakamura, Y., White, R., Smits, A. M. M. & Bos, J. L. (1988) N. Engl. J. Med. 319, 525-532. 4. Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R., Preisinger, A. C., Jessup, J. M., vanTuinen, P., Ledbetter, D. H., Barker, D. F., Nakamura, Y., White, R. & Vogelstein, B. (1989) Science 244, 217-221. 5. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R., Cleary, K., Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., Glover, T., Collins, F. S., Weston, A., Modali, R., Harris, C. C. & Vogelstein, B. (1989) Nature (London) 342, 705-708. 6. Takahashi, T., Nau, M. M., Chiba, I., Birrer, M. J., Rosenberg, R. K., Vinocour, M., Levitt, M., Pass, H., Gazdar, A. F. & Minna, J. D. (1989) Science 246, 491-494.
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