Detection of a rare point mutation in Ki-ras of a human bladder cancer xenograft by polymerase chain reaction and direct sequencing.
This paper represents the first report of a codon 59 mutation in Ki-ras from a spontaneous human transitional cell carcinoma of the bladder. Point mutations have the potential to activate the ras genes if they occur in critical coding regions. These include the sequences of codons 12, 13, 59, 61 and 63. Mutations in codons 12, 13 and 61 have been reported in a wide variety of human cancers, including transitional cell carcinoma of the bladder. However mutations in codon 59 have been reported only in retroviral Ki-ras and as a result of in vitro mutagenesis experiments. We have used the polymerase chain reaction and direct sequencing to detect mutations of Ki-ras, and allele-specific restriction analysis to detect mutations of N-ras in xenografts and continuous cell lines established from bladder cancer biopsies of ten different patients as well as in direct biopsy specimens from five human bladder tumours. For studies of Ki-ras, a 139 bp fragment which spanned the critical codons 12 and 13 and a 128 bp fragment that spanned the sequences of codon 59, 61 and 63 were enzymatically amplified and then sequenced. No N-ras mutations were detected. A heterozygous mutation of Ki-ras at codon 59 GCA----G/ACA was detected in one line. This mutation is being expressed and appears stable as it was detected over several xenograft passages and was present in paraffin-embedded tissue from the primary tumour of the patient. The biological significance of the mutation in bladder cancer is currently under study.
Grimmond
S M
SM
Urological Cancer Research Unit, Royal Prince Alfred Hospital, Sydney, Australia.
Raghavan
D
D
Russell
P J
PJ
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002295:Carcinoma, Transitional Cell / Q000235:genetics*; D003062:Codon / Q000235:genetics*; D004252:DNA Mutational Analysis; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
D003062:Codon