[CANCER RESEARCH 53. 1162-1166, March I. 1993] Distinct Pattern of p53 Mutations in Bladder Cancer: Relationship to Tobacco Usage1 Charles H. Spruck III, William M. Rideout III, Aria F. Oliimi, Petra F. Ohneseit, Allen S. Yang, Yvonne C. Tsai, Peter W. Nichols, Thomas Horn, Gregers G. Hermann, Kenneth Steven, Ronald K. Ross, Mimi C. Yu, and Peter A. Jones2 Kenneth Harris Jr. Comprehensive Cancer Center. Urologie Cancer Research Laboratory, University of Southern California. Los Angeles. California 90033 ¡C.H. S., W. M. K.. A. P.O.. P. E O.. A. S. Y.. Y. C. T.. P. W. N., K. K. R.. M. C. Y.. P. A. }.}, and Herlev Hospital. University of Copenhagen. DK-2730 Herlev. Denmark ¡T. H.. G. C. H., K. S.¡ ABSTRACT A distinct mutational spectrum for the p53 tumor suppressor gene in bladder carcinomas was established in patients with known exposures to cigarette smoke. Single-strand conformational polymorphism analysis of exons 5 through 8 of the p53 gene showed inactivating mutations in 16 of 40 (40%) bladder tumors from smokers and 13 of 40 (33%) tumors from lifetime nonsmokers. Overall, 13 of the 50 (26%) total point mutations discovered in this and previous work were G:C >C:G transversions, a relatively rare mutational type in human tumors. In six tumors, identical AGA (Arg) —¿> ACÕ (Thr) point mutations at codon 280 were observed, suggesting a mutational hotspot in these tumors. Comparison of the mu tational spectra from smokers and nonsmokers revealed no obvious dif ferences in the types or positions of inactivating mutations; however, 5 of 15 tumors containing point mutations from cigarette smokers had double mutations, four of which were tandem mutations on the same alÃ-ele.No double mutations were found in tumors from nonsmoking patients. None of the mutations in smokers were G:C •¿ I: A transversions, which would be anticipated for exposure to the suspected cigarette smoke carcinogen 4-aminobiphenyl. The results suggest that, although cigarette smoke ex posure may not significantly alter the kinds of mutations sustained in the p53 gene, it may act to increase the extent of DNA damage per mutagenic event. INTRODUCTION Carcinogenesis involves the accumulation of alterations in genes which function to regulate cellular growth. Both endogenous mu tagenic processes and exogenous factors, such as the direct or indirect effects of chemical carcinogens, are believed to induce DNA damage. Various studies have shown that chemical carcinogens can selectively induce specific base changes in cancer-related genes in vivo. For example, the Ha-ra.v oncogene has been shown to acquire specifically G —¿Â» A transitions in mammary tumors from rats treated with ni- trosomethylurea ( 1), while the same gene harbors A —¿> T transver sions in skin tumors from mice induced by dimethylbenzanthracene (2). Therefore, a carcinogenic agent may be identified by analysis of the pattern of mutations acquired in cancer-related genes during car- cinogenesis (3). The p53 tumor suppressor gene is ideally suited for molecular epidemiological studies. The gene product likely functions as a tran scription factor (4) and may be involved in the cellular response to DNA damage (5). Inactivation of the gene by mutation occurs in a diverse variety of human tumors over a large region of the gene (6). Several recent studies have associated preferential mutations in the p53 gene with specific carcinogenic agents. For example, selective Received 9/30/92; accepted 12/22/92. 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 by USPHS Grants ROI CA40468 and R35 CA49758 from the National Cancer Institute, by the Betty Lou Warren Research Fund, and by the California Tobacco- related Disease Research Program Grant 1RT423. 2 To whom requests for reprints should be addressed, at the Kenneth Norris Jr. Com prehensive Cancer Center, University of Southern California, 1441 Eastlake Avenue. Los Angeles, CA 90033-0800. G —¿> T transversions at codon 249 have been demonstrated in hepa- tocellular carcinomas in geographic regions where aflatoxin is a known risk factor (7, 8). In skin tumors, a prevalence of C —¿> T and CC —¿> TT mutations have been observed, presumably due to the phys ical damage induced on DNA by UV light (9). Furthermore, lung cancers contain a high percentage of G —¿> T transversions, a muta tional type known to be induced by benzo(a)pyrene, a constituent of cigarette smoke (10). Nucleotides in the p53 gene which undergo these mutations in lung tumors have been shown to be specifically targeted by activated benzo(a)pyrene metabolites in vitro (11). Bladder carcinoma, the fifth most common cancer in the United States, accounting for 5% of all tumors diagnosed, has an annual incidence of 49,000 new cases each year (12). Cigarette smoking has been shown to be a major risk factor for bladder cancer among men in the United States (13). Current cigarette smokers demonstrate a 2- to 3-fold elevation in relative risk of developing bladder cancer com pared with persons who do not use tobacco. We have previously shown that LOH3 of chromosome 17p, where the p53 gene resides, is a frequent event in high-grade bladder carcinomas (14). The remain ing p53 alÃ-eleis often inactivated by point mutation (15), and these mutations have been associated with tumor invasiveness (16). In the present study, the p53 gene was assayed for mutations in bladder carcinomas from patients who were current cigarette smokers, lifelong nonusers of tobacco products, or ex-smokers to determine the influ ence of carcinogens in cigarettes on the mutational spectra in bladder cancer. MATERIALS AND METHODS Patient Selection and DNA Extraction. Eighty bladder carcinoma speci mens (45 fresh-frozen and 35 paraffin-embedded tissues) were obtained from patients diagnosed in hospitals in Los Angeles County, CA (n = 62). and from the Herlev Hospital in Copenhagen, Denmark (n = 18). Tumors analyzed were histopathologically classified as grade III-IV transitional cell carcinomas, squamous cell carcinomas, or neuroendocrine differentiated carcinomas of the bladder according to the criteria of Ash (17) and Bergkvist et ai. ( 18). History of smoking was obtained through in-person interviews (n = 19), from medical records (n = 51), or through interviews with spouses (n = 10). Smokers were classified in most cases as patients with a defined duration and exposure of cigarette use and were currently smoking at the time of diagnosis (n = 40). Patients who had an exposure of <100 cigarettes during their lifetime and never used any additional tobacco-related products were classified as non- smokers (n = 40). Patients who had terminated cigarette use at least 7 years prior to cancer diagnosis were classified as ex-smokers (n = 4). High molecular weight DNA was prepared from fresh tumor specimens and matching blood samples by proteinase K digestion and phenol/chloroform extraction as described previously (19, 20). DNA was isolated from archival paraffin-embedded tissue samples by dissecting a 10-um cryostat section with a sterile scalpel to enrich for neoplastic cells. The sections were resuspended ' The abbreviations used are: LOH, loss of heterozygosity: SSCP, single-strand con formation polymorphism; 4-ABP, 4-aminobiphenyl; PCR, polymerase chain reaction; Arg, arginine; Lys. lysine; Glu, glutamic acid; Thr, threonine; Gin, glutamine; His, histidine; Gly, glycine. Ser, serine; Cys, cysteine; Tyr, tyrosine; Trp, trytophan; Pro. proline; Asn, asparagine; Ala, alanine; Term, termination. 1162 Research. on February 13, 2016. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from