p 53 Mutation Arising in Arg 72 Allele in the Tumorigenesis and Development of Carcinoma of the Urinary Tract 1

Purpose: It is known that a common p53 polymorphism, encoding either proline (Pro) or arginine (Arg) at residue 72, produces marked change in the structure of p53. Furthermore, the Arg72-containing allele is preferentially mutated and retained in various human tumors, suggesting that polymorphic residue within p53 modifies mutant behavior. We studied to determine whether Arg72 could be a risk factor for p53 mutations in human transitional cell carcinomas (TCCs). In addition, the relationship between the status of p53 codon 72 polymorphism and clinicopathological factors of this tumor were also analyzed. Experimental Design: We analyzed the correlation between the p53 mutations and genotypes of its codon 72 using genomic DNAs from the TCCs by direct DNA sequencing. Loss of heterozygosity was determined using a p53 microsatellite marker (TP53) amplified by PCR. Results: There was a bias to mutate and express the Arg allele in the p53-mutated TCCs arising in individuals with heterozygosity (Pro/Arg). The Arg72-containing allele was preferentially retained in these tumors. The prevalence of cases with p53 mutations within Arg72-containing allele was higher for advanced-stage TCCs ( 2 5.320, P 0.021) than for TCCs with those arising in Pro72-containing allele. Conclusions: Our in vivo findings suggested that p53 mutation alleles containing Arg72 are preferentially selected during tumorigenesis and affect mutant behavior in TCCs, and revealed that TCCs with p53 mutation arising in Arg72containing allele became progressively more abundant with increase in tumor stage. INTRODUCTION The tumor-suppressive functions of p53 stem, in part, from its capacity to induce cell cycle arrest in late G1 and/or apoptosis in response to genotoxic stress and hypoxia, and mutational inactivation of p53 is associated with increased risk of tumorigenesis (1). Oncogenic studies have revealed that missense mutations of the p53 gene lead to critical structural changes in the p53 product and are among the most common genetic alterations in human cancers. It is known that a p53 sequence polymorphism found in the general population that results in either Arg or Pro, or at residue 72 produces marked change in the structure of p53 protein and thereby affects migration on SDS-PAGE (2). Several reports have noted epidemiological differences in the prevalence or prognostic significance of this p53 polymorphism in certain cancer types (3–7). Recent reports suggested that the allele encoding Arg72 is a significant risk factor for the development of cervical and esophageal cancers associated with human papillomavirus (8–10). Two recent reports additionally indicated that the Arg72 allele is preferentially mutated and retained in various human tumors (11, 12). However, the biopathological significance of these variants of p53 polymorphism is unclear. To determine whether a common polymorphic residue of Arg72 within p53 could represent a risk factor for mutations in p53, we analyzed the relationship between p53 mutations and genotypes of its codon 72 in human TCCs of the urinary tract. We next determined the identity of the deleted allele in the TCCs arising in individuals with Pro/Arg heterozygotes that had undergone LOH at the TP53 locus. From these data, the relationship with various clinical and pathological factors were also analyzed. MATERIALS AND METHODS Tumor Tissue Samples and Patient Population. Tumor tissue specimens were obtained from 141 cases of human TCCs of the renal pelvis, ureter, and urinary bladder, which were obtained by nephroureterectomy (58 cases), radical or partial cystectomy (51 cases), and transurethral resection (49 cases) between 1998 and 2002 at the Department of Urology of Kochi Medical School and the Divisions of Urology of both Kochi Takasu Hospital and Fujisaki Hospital. Cases with preoperative radiation or chemotherapy were not selected. The patients included 106 men and 35 women. The median age of the patients was 69.4 years (range 55–86). The Institutional Review Committee for clinical investigation reviewed and approved the collection of pairs of tumor and corresponding normal mucosa or peripheral blood tissue samples for genetic analysis. Paired normal and tumor tissues were processed in a similar fashion at all three of the institutions from which genomic DNA was isolated. Received 11/26/01; revised 2/4/02; accepted 2/11/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. 2 To whom requests for reprints should be addressed, at Department of Pathology II, Kochi Medical School, Nankoku, Kochi 783-8505, Japan. Fax: 088-880-2336; E-mail: furihata@kochi-ms.ac. jp. 3 The abbreviations used are: Pro, proline; Arg, arginine; TCC, transitional cell carcinoma; LOH, loss of heterozygosity. 1192 Vol. 8, 1192–1195, May 2002 Clinical Cancer Research Research. on November 1, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals. org Downloaded from Analyses of p53 Mutations, Codon 72 Polymorphism, and LOH. Genomic DNAs isolated from TCCs have been analyzed by direct DNA sequencing concentrating on exons 4–9 of the p53 gene according to our procedure described previously (13). Primers were prepared to amplify exons 4–9, including each splicing site, of the p53 gene for direct DNA sequencing analysis. The sequence of each set of primers for PCR and the conditions of sequencing analysis of p53 were as described previously (13). Using genomic DNAs from each normal tissue, the identity of the amino acid at codon 72 in exon 4 of p53, Pro (CCC) or Arg (CGC), was also determined by the sequencing. In the determination of genotype of codon 72 polymorphism, the Pro or Arg type was scored if the area under the guanine peak was reduced to 50% of its cytosine allele (this indicate Pro type) or if the area under the cytosine peak was reduced to 50% of its guanine area (this indicate Arg type). For each primer pair, sense primer only was fluorescently labeled at the 5 end. Template genomic DNAs were amplified by PCR using a thermal cycler (Takara Co Ltd., Ohtsu, Japan) with Taq polymerase (Perkin-Elmer, Norwalk, CT). Direct genomic DNA sequencing of PCR products was performed using Automated Laser Fluorescent ALF sequencer (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) on a 6% polyacrylamide denaturing gel. In the cases that detected p53 mutation showing heterozygosity (Pro/Arg) at codon 72, genomic DNA fragments from the tumors containing exon 4 to each mutated exon detected were amplified by PCR, successively subcloned into pGEM-T easy plasmids (Promega, Madison, WI), and transfected into JH109 cells to identify the codon 72 genotype of the mutant allele of p53 (14). At least five JH109 clones of each case were expanded and subjected to sequence analysis. LOH was determined using a p53 microsatellite marker (TP53) amplified by PCR as described previously (15). LOH was defined as a reproducible reduction of 30% of either the smaller or larger allele. Samples that were homozygous for the marker were defined as noninformative and excluded from calculations for LOH frequency. Statistical Analysis. The 2 test at the 5% level was used to determine statistical associations between p53 mutations and the status of polymorphisms of codon 72. Analysis of associations with clinicopathological factors was also performed using the 2 test.