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Technical Briefs |
1
University of Vienna, Department of Obstetrics and Gynecology, Division of Gynecology, at the General Hospital of Vienna, A-1090 Vienna, Austria;
2
Ludwig-Boltzmann Institute for Gynecological Oncology and Reproductive Medicine, A-1090 Vienna, Austria;
a address
correspondence to this author at: University of Vienna, Department of Obstetrics and Gynecology, Molecular Oncology Group, Waehringer Guertel 18-20, EBO 05, A-1090 Vienna, Austria
The p53 tumor suppressor gene lies on chromosome 17p and encodes a nuclear phosphoprotein. Wild-type p53 protein can suppress cellular transformation and proliferation (1)(2). Mutations in the p53 gene are the most common genetic alteration in human cancers (3). More than one-half of all human cancers show either absence of the p53 protein function or mutations in the gene. Mutant and wild-type p53 may oligomerize, thereby disrupting the function of the tetrameric complex (4). Codon 72 of the p53 gene was found to be a site of frequent polymorphism (5). The frequencies of allelic variants at this codon not only differ among different ethnic groups (6)(7), they are also associated with cancer susceptibility (8)(9)(10).
A relationship between viral infection and cancer has been found. It has been reported that adenovirus E1b 55-kDa protein and human papillomavirus E6 protein can bind to and inactivate p53 protein (11)(12). Targeting of p53 by these viruses prevents cell death and induces the cellular proliferation that is necessary for viral replication. The development of most cervical carcinomas has been shown to be associated with human papillomavirus (HPV) types 16 and 18. The E6 oncoprotein encoded by HPV types 16 and 18 has been found to promote the degradation of p53 (13). Recently, it was suggested that the arginine form at codon 72 of p53 was more susceptible to E6-mediated degradation than the proline form. Individuals homozygous for the arginine form at codon 72 were approximately sevenfold more susceptible to HPV-associated tumorigenesis than heterozygotes (14).
We established a PCR and microtiter plate-based hybridization assay for the detection of the codon 72 polymorphism in p53. The system was evaluated with DNA from cell lines with known allelic information. We examined DNA from 105 cervical carcinoma patients and 133 healthy female volunteers, who were representative for the Austrian population.
EDTA-blood samples from 105 cervical carcinoma patients and 133 healthy volunteers were collected at the Department of Obstetrics and Gynecology, University of Vienna, from 1984 to 1998. The median age of the cervical carcinoma patients was 49 years (range, 25–92 years), whereas the median age of the control group was 54 years (range, 21–77 years). In addition, all patients were matched for ethnic background and represented the typical Austrian population. Informed consent was obtained from all patients, and all procedures were approved by the institute’s responsible committee.
Breast cancer cell lines T-47D, BT-549, BT-483, CAMA-1, DU4475, Hs 578.T, SK-BR-3, and MCF-7, and ovarian cancer cell lines OVCAR-3 and ES-2 were purchased from American Type Culture Collection. All cell lines were cultured according to the instructions from American Type Culture Collection.
DNA was isolated from blood and cell lines by commercially available kits (DNA Extraction Systems I and II; ViennaLab).
Sense primer 5'-ATGGATGATTTGATGCTGTC-3' and antisense primer 5'-AGAAGCCCAGACGGAAAC-3' were used for the amplification of a DNA fragment containing the polymorphic site at codon 72. The antisense primer was labeled with fluorescein. For PCR, 30 ng of DNA was used as template in a total volume of 30 µL. The reaction mixture included 15 pmol of both sense and antisense primers, 250 µmol/L dNTPs (ViennaLab), 3.0 µL of 10x amplification buffer (10 mmol/L Tris-HCl, pH 9.0, 50 mmol/L KCl, 0.1 g/L gelatin, 1.5 mmol/L MgCl2, 1.0 mL/L Triton X-100; ViennaLab) and 1.0 U of Super Taq Polymerase (HT Biotechnology). PCR was performed on a Perkin-Elmer GeneAmp PCR system 9600 with 40 cycles at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s. All reactions were preceded by a primary denaturation step at 94 °C for 1 min. PCR product (5 µL) was then resolved on 3% agarose gels containing 0.1 mL/L SYBR Green I (Molecular Probes). Gels were excited with 254 nm transilumination.
Oligonucleotides were designed to specifically hybridize either with PCR products from the p53 Pro72 isoform (5'-GCTCCCCCCGTGGC-3') or the Arg72 isoform (5'-CTGCTCCCCGCGTG-3'). The probes were 5' labeled with biotin. The detection of the p53 polymorphism was carried out using the Universal Gene Mutation Detection Kit (ViennaLab). Briefly, 10 µL of PCR product was denatured in an alkaline buffer and added to 100 µL of Tris-based hybridization buffer containing 5 pmol of specific oligonucleotide in a streptavidin-coated well. Hybridization was carried out at 37 °C for 30 min. After two stringent washes in a buffer containing 0.4x standard saline citrate at 37 °C, an anti-fluorescein horseradish peroxidase complex was added to the well. Then it was incubated at room temperature for 15 min. The well was washed twice with phosphate-buffered saline, and a color developer containing 3,3',5,5'-tetramethylbenzidine was added to the well. After 15 min of color development, an acidic buffer was added to stop the reaction, and the color intensity was measured at 450 nm with 630 nm as reference.
For system evaluation and to verify the results from microtiter well-based hybridization, PCR products from cell lines and from 20 randomly selected samples were sequenced directly using an ALFexpress DNA sequencer and the Thermo Sequenase fluorescently labeled primer cycle sequencing kit (Amersham Pharmacia Biotech).
Differences in the allelic frequencies of p53 at codon 72
between the cervical carcinomas and the control group as well as
between the Austrian population and other Caucasian populations were
examined by the 
2 test.
DNAs from several breast and ovarian cancer cell lines were used to
evaluate our detection system. Allelic information of p53 at
codon 72 in these cell lines was obtained from sequencing. As shown in
Fig. 1
A, after amplification of template DNAs by PCR, a 223-bp PCR
product was generated. Hybridization of these PCR products to the
oligonucleotides specific for the p53
Pro72 isoform and the Arg72
isoform on microtiter plates generated either a blue-colored positive
signal or a colorless negative signal in the wells. Fig. 1A
shows that
the cell lines T-47D, BT-549, and ES-2 are homozygous for
p53 Pro72, cell lines BT-483, CAMA-1,
Hs 578.T, SK-BR-3, and OVCAR-3 are homozygous for
Arg72, and the cell line MCF-7 is heterozygous
for Pro72 and Arg72. This
result was in accordance with the results from direct sequencing,
confirming that the detection of p53 polymorphism at codon
72 by PCR and allele-specific hybridization on microtiter plates is a
reliable method. The blue-colored positive signal changes to yellow
after addition of the acidic buffer. Positive signals usually have an
absorbance reading of >0.500, and negative signals are below 0.050 at
450 nm.
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To test the binding specificity of the allele-specific oligonucleotides
to the PCR products, different amounts of PCR products from DNAs with
different allelic types were applied to the hybridization system. Fig. 1B
shows that binding of the oligonucleotides to the corresponding PCR
products is specific. As the intensity of the signals increased with
the increasing amounts of PCR products, the intensities of the
unspecific binding were unchanged.
Analysis of the p53 polymorphism at codon 72 showed that the
proportion of individuals homozygous for arginine, homozygous for
proline, and heterozygous for arginine and proline were 54.3%, 7.6%,
and 38.1% among the 105 cervical carcinoma patients and 62.4%, 8.3%,
and 29.3% among the 133 healthy women (Table 1
). 2 analysis revealed no significant
differences in these proportions between the two groups.
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Sequencing of the PCR products from 20 randomly selected samples showed 100% concordance with the results obtained from PCR and allele-specific oligonucleotide hybridization on microtiter plates (data not shown).
In previous studies on the p53 polymorphism at codon 72, tumor samples were used as the source for DNA (14)(15)(16). Although loss of heterozygosity at the p53 locus is not common in cervical carcinomas, in one study, an allelic imbalance was observed in 22% of the samples (17). Another study demonstrated a loss of heterozygosity rate of 15% on chromosome 17p in cervical carcinomas; the authors suggested that this might be caused by alterations of the p53 gene (18). The use of DNA from tumor samples for polymorphism analysis may, therefore, lead to inaccurate results. In this study, we used DNA from peripheral blood lymphocytes because they are not affected by such genetic alterations frequently observed in solid tumors.
Comparing the frequencies of allelic variants found in Austrian women
with those reported in other Caucasian women (Table 1
)
(7)(16), both the control group and the
carcinoma patients showed no significant differences from the other
study groups, indicating that the frequencies of p53
variants at codon 72 are not different in Caucasian women in Sweden,
the United Kingdom, and Austria.
Our results show that there is no difference in the allelic frequencies of the p53 gene at codon 72 between cervical carcinoma patients and the control group. Because we did not determine HPV status in our study group, we cannot directly compare our results with the data of Storey et al. (14). Storey et al. reported that individuals homozygous for the arginine form at codon 72 are approximately sevenfold more susceptible to HPV-associated cervical cancer than heterozygotes. However, our data are in accordance with the results of two other studies (15)(16), one of which included only cases that were positive for HPV types 16 and 18. Both studies reported a lack of correlation between polymorphism at codon 72 of p53 and risk of cervical cancer. It cannot be ruled out that such a correlation exists in ethnic groups different from the ones investigated in these studies and ours because a significant difference for the prevalence of arginine homozygotes among various ethnic groups has been reported (6). Therefore, more epidemiological studies that include patients with different ethnic backgrounds should be undertaken. Detection of this p53 polymorphism with PCR and allele-specific hybridization on microtiter plates is amenable to automation and thus can be very useful for this purpose.
Acknowledgments
This study was supported by the Anniversary Fund of the Austrian National Bank for the Promotion of Scientific Research and Teaching (Project ÖNB 6054). We thank Andrea Wolf and Sabrina Zeillinger for isolating the DNA used in this study.
Footnotes
fax 43-1-40400-7832, e-mail robert.zeillinger{at}akh-wien.ac.at
References
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