Lack of Mutation in Exon 10 of P53 Gene in Thyroid Tumors I N V E S T I G a C I Ó N

T he tumor suppressor gene p53 is a transcription factor that acts in cell cycle regulation, inducing cell cycle arrest or cell death in response to DNA-damaging agents, such as viral infection, radiation and chemotherapeutics 1. The p53 protein resides primarily in the nucleus, binds to specific DNA sequences, and functions at least in part as a transcriptional regulator 2. Inactivated p53 mutations have been described in some 50% of human cancers and are believed to be a major determinant of the phenotype of many forms of cancer 1-3. Several studies, both with immunocytochemical and genetic analyses, have shown that p53 mutations are highly prevalent in poorly differentiated and undifferentiated thyroid carcinomas, as well as thyroid cancer cell lines 4-6. However, they are not found in benign tumors and are infrequent in well-differentiated cancers, suggesting that mutational inactivation of p53 occurs at a late stage of thyroid tumor progression 7. These data suggest that mutatio-nal inactivation of the p53 gene may be a key event in the progression from differentiated to anaplastic carcinoma 4,7. There is also evidence that p53 may interfere with thyroid cell differentiation. Introduction of a mutated p53 markedly impairs the differen

T he tumor suppressor gene p53 is a transcrip- tion factor that acts in cell cycle regulation, inducing cell cycle arrest or cell death in response to DNA-damaging agents, such as viral infection, radiation and chemotherapeutics 1 .The p53 protein resides primarily in the nucleus, binds to specific DNA sequences, and functions at least in part as a transcriptional regulator 2 .Inactivated p53 mutations have been described in some 50% of human cancers and are believed to be a major determinant of the phenotype of many forms of cancer [1][2][3] .
Several studies, both with immunocytochemical and genetic analyses, have shown that p53 mutations are highly prevalent in poorly differentiated and undifferentiated thyroid carcinomas, as well as thyroid cancer cell lines [4][5][6] .However, they are not found in benign tumors and are infrequent in welldifferentiated cancers, suggesting that mutational inactivation of p53 occurs at a late stage of thyroid tumor progression 7 .These data suggest that mutational inactivation of the p53 gene may be a key event in the progression from differentiated to anaplastic carcinoma 4,7 .There is also evidence that p53 may interfere with thyroid cell differentiation.Introduction of a mutated p53 markedly impairs the differen- tiated gene expression of PCC13 thyroid cells 8 .By contrast, wild-type p53 reintroduction into an undifferentiated thyroid carcinoma cell line leads to reexpression of thyroid peroxidase, a characteristic differentiated marker of the thyroid cell 9 .
Typically, mutations in p53 gene are located in exons 5-8, a highly conserved DNA binding domain of p53.Recently, a distinct nucleotide substitution in the exon 10 of p53 was identified at a high frequency, 77 to 97% of children with benign and malignant adrenocortical sporadic tumors investigated by 2 distinct groups 10,11 .This germline mutation leading to an Arg337His mutation of exon 10 was also identified in asymptomatic relatives of the patients but in none of the unrelated controls, suggesting that the mutation is a risk factor associated with adrenocortical tumors rather than a benign polymorphism commonly found in southern Brazil 10,11 .
Sporadic tumors often appear to have the same gene mutations as their familial counterparts.Many germline mutations have been demonstrated to be associated with sporadic tumors, including thyroid cancer [12][13][14][15][16] .We recently showed that a polymorphism at codon 72 of exon 4 of p53 was associated with sporadic thyroid carcinomas 17 .
Because of the high prevalence of the codon 337 of exon 10 of p53 mutation in southern Brazilian population and the possibility that this polymorphism could be also associated to other cancers, we designed this study to screen a large amount of samples for this p53 mutation in thyroid tumors.

Subjects. The Ethics Committee of the University
Hospital -School of Medicine of the State University of Campinas (HC-FCM/UNICAMP) approved the study and informed written consent was obtained from a total of 74 subjects (55 females, 19 males, 16 to 81 years old, 49±21 years old) that were consecutively referred to thyroid surgery because of thyroid nodules that presented clinical or epidemiological suspicion of cancer.The diagnosis of thyroid carcinoma was established by fine-needle aspiration cytological study and confirmed by the histological analysis of thyroid tissues.There were 28 thyroid malignant tumors: 5 follicular carcinomas (3 widely invasive and 2 minimally invasive); 22 papillary carcinomas (14 of the classic variant, 2 follicular variants, 6 tall cell variants) and 1 medu-llary carcinoma.Other 46 cases (35 females, 11 males, 21 to 75 years old, 47±19 years old) of benign goitres included 19 follicular adenomas, 22 multinodular goitres and 5 Basedow-Graves disease.Thyroid tissue samples were obtained at the time of surgery at the University Hospital and immediately frozen in liquid N2.Besides collecting a central portion of all tumors, we obtained samples from the contra lateral normal thyroid lobe of 26 patients with thyroid cancer.In addition, peripheral blood samples were collected from 18 different patients with benign goitres.Tumor stage and degree of differentiation were obtained from surgical and pathological records.Experienced pathologists of the University Hospital of the Faculty of Medical Sciences of the State University of Campinas (UNICAMP) confirmed all diagnoses.
Methods.Genomic DNA was extracted from frozen tumors using a standard phenol-chloroform method.We used the same primers described by Latronico et al 10 .PCR was performed in 25 µl volumes of a mixture containing 100 ng DNA, 50 nM of each primer (5'-CTGAGGCACAAGAATCAC-3' and 5'-TCC-TATGGCTTTCCAACC-3'), 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 100 uM of each dinucleotide triphosphate and 0.5 U Taq DNA polymerase.Amplifications were carried out for 35 cycles of 94°C for 45 seconds, 62°C for 45 seconds and 72°C for 1 min, with an initial denaturation step of 94°C for 2 min and a final extension step of 72°C for 7 min using a Perkin-Elmer 9600 GeneAMp PCR system.The amplified 447 bp DNA fragments were examined on a 2% agarose gel, containing ethidium bromide.After confirming amplification, the samples were mixed with 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol and 50 mM NaOH, denatured at 94°C for 10 min, and loaded on to 6% polyacrylamide gels.The electrophoresis was conducted at 2-5 W at room temperature overnight.The gel was then stained with silver nitrate.DNA samples homo and heterozygous for the Arg337His mutation, obtained from adrenocortical tumors, were used as positive controls of the gels.

RESULTS
Figure 1 depicts an example of our results.All samples showed the same pattern of running, with no significant differences.Two samples suspected of presenting aberrant migrating bands were exci- sed from the gel and purified using a commercial kit according to the manufacturer's instructions (Life Technologies, Paisley, UK).PCR products were sequenced with the ABI prism big dye sequencing kit (Perkin Elmer, Warrington, Cheshire, UK) using an ABI 377 Prism DNA Sequencer (Perkin Elmer).In all cases a wild-type sequence was found.In addition, we directly sequenced 3 additional PCR products from tumor samples with normal SSCP patterns, and all were wild type.

DISCUSSION
The p53 gene is one of the best studied tumor suppressor genes, located on chromosome 17p13.1.Its mutation has been reported mainly in aggressive forms of tumors, especially anaplastic carcinomas 18 .It has been found in up to 40% of dedifferentiated and undifferentiated thyroid carcinomas and in less than 10% of the differentiated thyroid tumors 19 .However, mutant p53 protein has also been detected in follicular and papillary carcinomas 20 .More recently, p53 mutant protein was also demonstrated in 11 out of 66 nodular hyperplasia cases (16.7%) and in 7 out of 50 (14%) cases of follicular adenomas 21 .
Although somatic mutations of p53 are the most common genetic changes observed to date, the frequency of germline p53 mutations is found to be very low in sporadic malignant tumors 4 .It has been postulated that de novo germline p53 mutations may occur in a substantial population of patients in the pediatric age group, who die of their disease and do not propagate the mutation 22,23 .On the other hand, recent reports suggest that germline p53 splicing mutations have been described infrequently in the literature because the method of mutation detection, in many studies, does not include all splice junctio-ns24.The low figures reported in the literature might also reflect the use of less-sensitive mutation detection methods and, certainly, the fact that most researches focused on exons 5-8, within the DNAbinding domain of p53, instead of screening all 11 exons of TP53 24 .Indeed, because 85% of p53 mutations are expected to occur in exons 5 through 8, thyroid tumor screening efforts, in almost all reports, were restricted to these regions of the gene (http://www.iarc.fr/p53/;http://cancergenetics.org/p53.htm).
The spectrum and frequency of cancers associated with germline p53 mutations are uncertain.Some cancers like breast carcinoma, soft tissue sarcomas, osteosarcoma, brain tumors, adrenocortical carcinoma, Wilms' tumor and phyllodes tumor are strongly associated with germline p53 mutations while carcinoma of pancreas is moderately associated and leukaemia and neuroblastoma are weakly associated 25 .
Screening exon 10 by PCR-SSCP and by direct sequencing, we did not find mutations in a large number of thyroid samples.These results support the concept that germline TP53 mutations do not simply increase general cancer risk.Instead, they promote tissue-specific effects.Although our results are constrained by the fact that we did not screen poorly differentiated or undifferentiated tumors, they suggest that the Arg337His germline mutation described in Brazilian children is restricted to adrenocortical tumors.

FIGURE 1 .
FIGURE 1. Gel of single-stranded conformation polymorphism analysis of PCR products (PCR-SSCP) representative of our results for exon 10 of p53 gene screening for mutations.Lanes 1 and 2 were loaded with the positive controls for the homo-and the heterozygous Arg337His mutation of exon 10 of the p53 gene, respectively.Lanes 3-7 and 8-12 were loaded with PCR products from follicular and papillary carcinomas, respectively.