Mutagenesis, Vol. 15, No. 1, 57-60,
January 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
p53 intron 7 polymorphisms in urinary bladder cancer patients and controls
Department of Biosciences at NOVUM, Karolinska Institutet, 141 57 Huddinge and 1 Clinical Epidemiology, Department of Oncology–Pathology, Karolinska Hospital, 171 76 Stockholm, Sweden
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Abstract |
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A C
T polymorphism in intron 7 of the human tumour suppressor gene p53 was studied in 159 urinary bladder cancer patients and 171 non-cancer controls. The polymorphism was found in 15% of both patients and controls, suggesting that it has no relevance in urinary bladder cancer pathogenesis or aetiology. A second polymorphism, a TG change located 20 bp downstream of the CT change, was found in all samples with the CT change. Our findings indicate that the CT and the TG changes occur simultaneously and belong to the same allelotype. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Inactivation of the human tumour suppressor gene p53 is a common genetic change in human cancers (Hainaut et al., 1998
). The protein encoded by the normal allele of p53 is a 53 kDa nuclear protein, 393 amino acids long, that has several biological functions. It activates transcription of a number of genes and is up-regulated in response to certain stimuli such as DNA damage, thereby inducing a cellular response resulting in cell growth arrest or apoptosis (reviewed in Steele et al., 1998). p53 mutations are found in >50% of human tumours (Hainaut et al., 1998). Amino acids 117–286, found within exons 5–8, contain four of the five evolutionarily conserved domains of the p53 gene and the majority of reported mutations are found within this area (Greenblatt et al., 1994). The correlation between p53 mutations and human cancers has been thoroughly studied. Less well established is the functional significance of p53 polymorphisms. p53 is a highly conserved gene and there are only three polymorphisms reported in the coding region, two in exon 4, residues 47 (Felley-Bosco et al., 1993) and 72 (Matlashewski et al., 1987), and one is in exon 6, residue 213 (Carbone et al., 1991). Several groups have studied the association of a p53 codon 72 Arg/Pro polymorphism and increased cancer susceptibility. Jin et al. (1995) and Wang et al. (1999) found an association between the Pro/Pro phenotype and lung cancer and Storey et al. (1998) found an association between the Arg/Arg phenotype and increased susceptibility to HPV-associated cancers, but this finding was not confirmed by other studies (Helland et al., 1998; Hildesheim et al., 1998; Josefsson et al., 1998; Minaguchi et al., 1998; Yamashita et al., 1999). p53 polymorphisms are also found in the intronic regions, two being reported in intron 1 (Buchman et al., 1988; Futreal et al., 1991), one in intron 2 (Oliva et al., 1995), one in intron 3 (Lazar et al., 1993), two in intron 6 (Chumakov and Jenkins, 1991; McDaniel et al., 1991) and two in intron 7 (Prosser and Condie, 1991).
We have studied the two documented polymorphisms in intron 7 and their association with urinary bladder cancer in 159 bladder cancer patients and 171 non-cancer controls. A CT polymorphism is located at position 14181, 72 bp downstream of the 3'-end of exon 7 of the human p53 gene (GenBank accession no. X54156) and is apparently linked to a second polymorphism in intron 7, a TG change, 20 bp further downstream at position 14201.
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Materials and methods |
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Tissue
DNA from tumour tissue and matched blood samples from 159 urinary bladder cancer patients was extracted as previously reported (Sambrook et al., 1989
; Wada et al., 1999). The control DNA came from 171 non-cancer Swedish males aged 22–61 years.
PCR conditions
PCR was performed in a total volume of 10 µl containing 20 ng genomic DNA, 3 pmol each of forward and reverse primers (Table I), 1x Tris–HCl PCR buffer, 2 mM MgCl2, 0.11 mM dNTPs, 10% glycerol and 0.5 U Platinum Taq polymerase (Life Technologies). PCR was carried out for 36 cycles, four cycles with the higher annealing temperature and 32 cycles with the lower. Annealing temperatures were 63/62°C for primer pair 1 and 56/55°C for primer pair 2. The PCR machine used was a DNA Engine Peltier Thermal Cycler (MJ Research).
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SSCP conditions
Samples of 1 µl of the PCR reactions from tumour samples and a template-free blank were denatured in 2 µl of formamide/MgCl2/blue dextran denaturing buffer at 95°C for 3 min and then loaded onto two different native gels. Gel 1 was made from a 0.6x MDE solution (FMC In vitro AB) with 5% glycerol and was run for 10 h without cooling (temperature 33°C). Gel 2 was made from a 0.5x MDE solution with 5% glycerol and 1 M urea and was run for 8 h, with cooling at 20°C. Gel electrophoresis was carried out on an ABI 377 automated sequencer (Perkin Elmer Applied Biosystems) with an external cooling system attached. Running parameters for both gels were 3.0 kV, 60 mA and 100 W using a 1x TBE buffer (pH 8.3).
Sequencing conditions
Fragments showing band shifts in SSCP analysis (Figure 1) were sequenced from new PCR reactions using DyeDeoxy terminator cycle sequencing. Both sense and antisense strands were sequenced from tumour samples; only forward strands were sequenced from corresponding normal tissue. Randomly chosen samples without band shifts in SSCP analysis were sequenced as negative controls for the polymorphism. PCRs for sequencing reactions were made in 50 µl volumes using the same conditions and primers as above. PCR reactions were purified using S-400 columns (Amersham-Pharmacia Biotech). For sequencing reactions either a Thermosequenase 2.0 sequencing kit (Amersham-Pharmacia Biotech) or a Big Dye sequencing kit (Perkin Elmer) was used according to the manufacturer's instructions.
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Restriction enzyme assay
A restriction enzyme assay was set up to test if the polymorphism was as frequent in control samples as in urinary bladder cancer patients. Since the polymorphism was located three bases upstream of the p53ex7reverse primer binding site (primer pair 1), a new primer pair was synthesized to avoid artefacts (primer pair 2). A total of 171 healthy controls were investigated for the polymorphism using PCR and the restriction enzyme Eco47I (an isoschizomer of AvaII) that specifically cleaves at 5'-G
GTCC-3'. Thus only samples with the polymorphism are cut, leaving two fragments of 180 and 61 bp, whereas the wild-type remains as one 241 bp long fragment. Samples with the polymorphism are easily detected on a 5% PAGE gel stained with ethidium bromide (Figure 2). All polymorphisms were sequenced to determine if they were homozygous or heterozygous.
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Results |
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We detected a C
T polymorphism at position 14181 of the p53 gene (Figure 3) in 24 of 159 urinary bladder cancer patients and in 26 of 171 controls. One tumour sample was homozygous TT but the normal tissue was heterozygous, suggesting a loss of heterozygosity in the tumour sample. One of the control samples was homozygous TT and one sample was not sequenced since there was not enough material. Genotype frequencies for the urinary bladder cancer patients were CT –0.151, TT –0.0, CC –0.849 and genotype frequencies for the controls were CT –0.146, TT –0.006, CC –0.848. These genotypes give a calculated allele frequency of C 0.92 and T 0.08 for both urinary bladder cancer patients and controls.
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All sequenced samples that were heterozygous C
T at position 14181 were also heterozygous for a TG polymorphism 20 bp further downstream at position 14201 (Figure 3
). The tumour sample and the control sample that were TT homozygous at 14181 were also GG homozygous at 14201, suggesting that the CT and the TG polymorphisms belong to the same allelotype. When sequencing for the polymorphism, we found a T instead of the reported wild-type G at position 14168. This was true, without ambiguity, for all sequenced samples, including DNA from control samples and from urinary bladder cancer samples, with and without band shifts. We therefore conclude that T is the wild-type base at this position.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study of over 300 samples, we are in the position of giving an accurate estimate of the prevalence of a C
T polymorphism at position 14181 of the p53 gene. The frequency of the polymorphism was 15% and was virtually identical between urinary bladder cancer patients and controls, indicating that the polymorphism has no bearing on urinary bladder cancer pathogenesis or aetiology. A weakness of the study is that urinary bladder cancer patients and population controls do not represent the same population, although both populations are from Sweden. This discrepancy influences the results if there is a variation in the prevalence of the polymorphism, e.g. in relation to geography, age or gender. We have no indication that such is the case. Finally, our data indicate that there is an apparently linked polymorphism, a TG change 20 bp downstream of the described polymorphism, at position 14201.
In a study by Prosser and Condie (1991) the CT polymorphism was found in six of 60 breast cancer patients and in three of 23 controls. In the sequenced samples with the CT polymorphism they also found a TG polymorphism 20 bp further downstream. We have analysed a total of 300 cases and controls which makes our material larger than any previously analysed material for these polymorphisms. Even though, due to primer design, a selection for the CT change was made so that only CT changes were sequenced and confirmed to have TG changes and not vice versa, it seems apparent from the size of our material that the two polymorphisms occur concurrently. In all 50 samples where we found the CT change the TG change was also found, including two samples that were both TT as well as GG homozygous, which further supports our hypothesis that the two polymorphisms belong to the same allelotype. The two polymorphisms in intron 7 probably result from the same mutational event. They are located only 20 bp apart and our data suggest complete linkage, therefore it is not likely that one polymorphism is a compensatory event for the other.
Other linked polymorphisms in tumour suppressor genes have previously been reported for the CDKN2A gene by Zhang et al. (1994) and Aitken et al. (1999), but without establishing any association with increased susceptibility to cancer. The genes encoding the cytochrome p450 metabolic enzymes are highly polymorphic and a number of reports have studied polymorphic variants in relation to increased cancer susceptibility (Rannug et al., 1995; Smith et al., 1998). In one of the genes, CYP1A1, there is an intragenic IleVal polymorphism that is closely linked to a 3'-flanking m1m2 polymorphism (Hayashi et al., 1991). An enhanced lung cancer risk has been reported for individuals homozygous for this rare allele of CYP1A1 by Kawajiri et al. (1990), whereas Hirvonen et al. (1992) found no such association. There is no reason to believe that future studies of the two polymorphisms in p53 intron 7 will help us to understand the pathogenesis or aetiology of urinary bladder cancer.
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Notes |
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2 To whom correspondence should be addressed. Tel: +46 8 608 92 38; Fax: +46 8 608 15 01; Email: petra.berggren{at}cnt.ki.se
3 The Stockholm Bladder Cancer Group consists of Jan Adolfsson, Eric Borgström, Johan Hansson, Ulf Norming, Gunnar Steineck and Hans Wijkström
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References |
|---|
|
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-
Aitken,J., Welch,J., Duffy,D., Milligan,A., Green,A., Martin,N. and Hayward,N. (1999) CDKN2A variants in a population-based sample of Queensland families with melanoma. J. Natl Cancer Inst., 91, 446–452.
Buchman,V.L., Chumakov,P.M., Ninkina,N.N, Samarina,O.P. and Georgiev,G.P. (1988) A variation in the structure of the protein-coding region of the human p53 gene. Gene, 70, 245–252.[Web of Science][Medline]
Carbone,D., Chiba,I. and Mitsudomi,T. (1991) Polymorphism at codon 213 within the p53 gene. Oncogene, 6, 1691–1692.[Web of Science][Medline]
Chumakov,P.M. and Jenkins,J.R. (1991) BstNI/NciI polymorphism of the human p53 gene (TP53). Nucleic Acids Res., 19, 6969.
Felley-Bosco,E., Weston,A., Cawley,H.M., Bennett,W.P. and Harris,C.C. (1993) Functional studies of a germ-line polymorphism at codon 47 within the p53 gene. Am. J. Hum. Genet., 53, 752–759.[Medline]
Futreal,P.A., Barrett,J.C. and Wiseman,R.W. (1991) An Alu polymorphism intragenic to the TP53 gene. Nucleic Acids Res., 19, 6977.
Greenblatt,M.S., Bennett,W.P., Hollstein,M. and Harris,C.C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54, 4855–4878.
Hainaut,P., Hernandez,T., Robinson,A., Rodriguez-Tome,P., Flores,T., Hollstein,M., Harris,C.C. and Montesano, R. (1998) IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools. Nucleic Acids Res., 26, 205–213.
Hayashi,S., Watanabe,J., Nakachi,K. and Kawajiri,K. (1991) Genetic linkage of lung cancer-associated MspI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P450IA1 gene. J.Biochem. (Tokyo), 110, 407–411.
Helland,A., Langerod,A., Johnsen,H., Olsen,A.O., Skovlund,E. and Borresen-Dale,A.L. (1998) p53 polymorphism and risk of cervical cancer [letter; comment]. Nature, 396, 530–531; discussion 532.[Medline]
Hildesheim,A., Schiffman,M., Brinton,L.A., Fraumeni,J.F., Jr., Herrero,R., Bratti,M.C., Schwartz,P., Mortel,R., Barnes,W., Greenberg,M., McGowan,L., Scott,D.R., Martin,M., Herrera,J.E. and Carrington,M. (1998) p53 polymorphism and risk of cervical cancer [letter; comment]. Nature, 396, 531–532.[Medline]
Hirvonen,A., Husgafvel-Pursiainen,K., Karjalainen,A., Anttila,S. and Vainio,H. (1992) Point-mutational MspI and Ile-Val polymorphisms closely linked in the CYP1A1 gene: lack of association with susceptibility to lung cancer in a Finnish study population. Cancer Epidemiol. Biomarkers Prev., 1, 485–489.[Abstract]
Jin,X., Wu,X., Roth,J.A., Amos,C.I., King,T.M., Branch,C., Honn,S.E. and Spitz,M.R., (1995) Higher lung cancer risk for younger African-Americans with the Pro/Pro p53 genotype. Carcinogenesis, 16, 2205–2208.
Josefsson,A.M., Magnusson,P.K., Ylitalo,N., Quarforth-Tubbin,P., Ponten,J., Adami,H.O. and Gyllensten,U.B. (1998) p53 polymorphism and risk of cervical cancer [letter; comment]. Nature, 396, 531; discussion 532.
Kawajiri,K., Nakachi,K., Imai,K., Yoshii,A., Shinoda,N. and Watanabe,J. (1990) Identification of genetically high risk individuals to lung cancer by DNA polymorphisms of the cytochrome P450IA1 gene. FEBS Lett., 263, 131–133.[Web of Science][Medline]
Lazar,V., Hazard,F., Bertin,F., Janin,N., Bellet,D. and Bressac,B. (1993) Simple sequence repeat polymorphism within the p53 gene. Oncogene, 8, 1703–1705.[Web of Science][Medline]
Matlashewski,G.J., Tuck,S., Pim,D., Lamb,P., Schneider,J. and Crawford,L.V. (1987) Primary structure polymorphism at amino acid residue 72 of human p53. Mol. Cell. Biol., 7, 961–963.
McDaniel,T., Carbone,D., Takahashi,T., Chumakov,P., Chang,E.H., Pirollo,K.F., Yin,J., Huang,Y. and Meltzer,S.J. (1991) The MspI polymorphism in intron 6 of p53 (TP53) detected by digestion of PCR products. Nucleic Acids Res., 19, 4796.
Minaguchi,T., Kanamori,Y., Matsushima,M., Yoshikawa,H., Taketani,Y. and Nakamura, N.(1998) No evidence of correlation between polymorphism at codon 72 of p53 and risk of cervical cancer in Japanese patients with human papillomavirus 16/18 infection. Cancer Res., 58, 4585–4586.
Oliva,M.R., Saez,G.T., Latres,E. and Cordon-Cardo,C. (1995) A new polymorphic site inintron 2 of TP53 characterizes LOH in human tumors by PCR-SSCP. Diagn. Mol. Pathol., 4, 54–58.[Web of Science][Medline]
Prosser,J. and Condie,A. (1991) Biallelic ApaI polymorphism of the human p53 gene(TP53). Nucleic Acids Res., 19, 4799.
Rannug,A., Alexandrie,A.K., Persson,I. and Ingelman-Sundberg,M. (1995) Genetic polymorphism of cytochromes P450 1A1, 2D6 and 2E1: regulation and toxicological significance. J. Occup. Environ. Med., 37, 25–36.[Web of Science][Medline]
Sambrook,J., Fritschookstein,E.F. and Maniatis,T. (1989) Molecular cloning. A laboratory manual, 2nd Edn. Cold Spring Harbour Press.
Smith,G., Stubbins,M.J., Harries,L.W. and Wolf,C.R. (1998) Molecular genetics of the human cytochrome P450 monooxygenase superfamily. Xenobiotica, 28, 1129–1165.[Web of Science][Medline]
Steele,R.J., Thompson,A.M., Hall,P.A. and Lane,D.P. (1998) The p53 tumour suppressor gene. Br. J. Surg., 85, 1460–1467.[Web of Science][Medline]
Storey,A., Thomas,M., Kalita,A., Harwood,C., Gardiol,D., Mantovani,F., Breuer,J., Leigh,I. M., Matlashewski,G. and Banks,L. (1998) Role of a p53 polymorphism in the development of human papillomavirus-associated cancer [see comments]. Nature, 393, 229–234.[Medline]
Wada,T., Louhelainen,J., Hemminki,K., Adolfsson,J., Wijkström,H., Norming,U. Borgström, E., Hansson,J. and Steineck,G. (1999) Bladder cancer; deletions at Rb and number of loci with deletions in 13q11 to 13q32, in relation to stage and grade. Clinical Cancer Research, in press.
Wang,Y.C., Chen,C.Y., Chen,S.K., Chang,Y.Y. and Lin,P. (1999) p53 codon 72 polymorphism in Taiwanese lung cancer patients: association with lung cancer susceptibility and prognosis. Clin. Cancer Res., 5, 129–134.
Yamashita,T., Yaginuma,Y., Saitoh,Y., Kawai,K., Kurakane,T., Hayashi,H. and Ishikawa,M. (1999) Codon 72 polymorphism of p53 as a risk factor for patients with human papillomavirus-associated squamous intraepithelial lesions and invasive cancer of the uterine cervix. Carcinogenesis, 20, 1733–1736.
Zhang,S.Y., Klein-Szanto,A.J.P., Sauter,E.R., Shafarenko,M., Mitsunaga,S., Nobori,T., Carson, D.A., Ridge,J.A. and Goodrow,T.L. (1994) Higher frequency of alterations in the p16/CDKN2gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck. Cancer Res., 54, 5050–5053.
Received on June 9, 1999; accepted on September 13, 1999.
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