title: Intragenic Tsc2 somatic mutations as Knudson's second hit in spontaneous and chemically induced renal carcinomas in the Eker rat model.
abstract: We searched for the rat homologue of the human tuberous sclerosis (TSC2) gene mutations in loss of heterozygosity (LOH)-negative Eker rat renal carcinomas (RCs) by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis using 45 primer sets covering all 41 coding exons and one leader exon including splicing donor/acceptor sites. We have identified intragenic somatic mutations in 7 of 21 spontaneous RCs, including one cell line (33%), and in 3 of 9 (33%) N-ethyl-N-nitrosourea (ENU)-induced LOH-negative RCs. Interestingly, five mutations in the spontaneous RCs were either deletion or duplication (5/7 = 71%). In contrast, all three in ENU-induced RCs were base substitutions (3/3 = 100%), as expected. Thus, a qualitative difference in the second hit might exist between spontaneous and ENU-induced mutations (e.g., deletion or duplication versus point mutation). By a direct cloning approach utilizing the restriction length difference caused by germline insertional mutation or reverse transcriptase-PCR analysis in two applicable cases, we could clearly show the presence of intragenic somatic mutations in the second copy (wild-type) of the Tsc2 gene. This is the first demonstration at the DNA sequence level of the validity of Knudson's two-hits hypothesis in the Tsc2 gene.
authors.affiliation: Department of Experimental Pathology, Cancer Institute, Tokyo.
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: S
authors.initials: S
authors.lastname: Urakami
authors.forename: Y
authors.initials: Y
authors.lastname: Hirayama
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: M
authors.initials: M
authors.lastname: Nishizawa
authors.forename: T
authors.initials: T
authors.lastname: Takahara
authors.forename: Y
authors.initials: Y
authors.lastname: Kubo
authors.forename: O
authors.initials: O
authors.lastname: Hino
mesh_terms: D000483:Alleles; D000818:Animals; D002872:Chromosome Deletion; D017931:DNA Primers; D004251:DNA Transposable Elements; D005038:Ethylnitrosourea; D016368:Frameshift Mutation; D016147:Genes, Tumor Suppressor; D006801:Humans; D007438:Introns; D007680:Kidney Neoplasms / Q000139:chemically induced / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D051381:Rats; D011919:Rats, Inbred Strains; D012097:Repressor Proteins / Q000235:genetics*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D012097:Repressor Proteins; C000624653:TSC2 protein, human; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D005038:Ethylnitrosourea