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Keywords:

  • p53;
  • bladder cancer;
  • N-butyl-N-(4-hydroxybutyl)nitrosamine;
  • N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide;
  • aminofluorene;
  • N-methylnitrosourea

Abstract

p53 genes were analyzed for mutations and expression in a series of 24 tumors or hyperplastic lesions of the urinary bladder induced in F344 rats by carcinogen treatment. Of these, 18 were analyzed as short-term urothelial cultures. Polymerase chain reaction–single-strand conformation polymorphism analysis and DNA sequencing were used to detect alterations in p53 genes or cDNAs, and the relative amounts of p53 protein per cell were estimated by immunohistochemical staining. Missense substitutions were found in the exon 5–9 region of two of five cell cultures analyzed from lesions induced by the bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. One of these was a papillary nodular hyperplasia, indicating that p53 mutations can be present in low- as well as high-stage/grade bladder lesions. p53 mutations were not found in the exon 5–9 region in cells of any of eight bladder lesions induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), including five transitional cell carcinomas (TCCs), or either of two TCCs induced by N-methylnitrosourea. Two of nine TCCs induced by the N-glucuronide of N-hydroxy-2-aminofluorene were found to have p53 mutations. One of these was evidently altered by three genetic events: a missense substitution in exon 8, a nonsense mutation in exon 6, and silencing of the “nonsense” allele (i.e., only the p53 missense mutation was detected). Immunohistochemical analysis with monoclonal antibody PAb240 (which preferentially binds to mutant p53 protein) detected p53 antigen only in those samples in which missense p53 mutations were found. With monoclonal antibody PAb421 (which detects mutant and wild-type p53), p53 antigen was also detected in cells from F542, a bladder tumor induced by FANFT in which no p53 mutations were found. Northern blot hybridization analysis showed that p53 transcripts were elevated twofold to threefold in several cases, including F542, suggesting that constitutive overexpression of wild-type p53 may occur in some bladder neoplasias. These data support the view that p53 may be involved in multiple rate-limiting steps in neoplastic transformation and may be a continuing target during bladder carcinogenesis. The data also contribute to evidence that certain chemical carcinogens may directly alter p53 genes during tumorigenesis. © 1994 Wiley-Liss, Inc.