Abstract
Activating ras mutations are found in many types of human tumour. Mutations in Harvey (H-), Kirsten (K-) and neuronal (N-) ras are, however, rarely found in breast carcinomas. TC21 is a ras family member that shares close homology to H-, K- and N-ras, and activating mutations have been found in ovarian carcinoma and leiomyosarcoma cell lines. We have examined panels of cDNAs from breast, ovarian and cervical cell lines, and primary and metastatic breast tumours for mutations in TC21 using a single-strand conformational polymorphism (SSCP)-based assay. One breast cancer cell line, CAL51, exhibited an altered SSCP pattern, compared with normal tissue, which was due to an A-T base change in codon 72, causing a predicted Gln-Leu activating mutation. Of nine primary and 15 metastatic breast tumour cDNAs analysed, none exhibited an altered pattern by SSCP. The apparently wild-type pattern by SSCP analysis was confirmed by sequence analysis of some of the cDNAs assayed. Thus, we conclude that mutations in TC21 are uncommon in breast carcinomas.
Highlights
K- and N-ras. and activating mutations have been found in ovarian carcinoma and leiomyosarcoma cell lines
We have examined panels of cDNAs from breast. ovarian and cervical cell lines, and primary and metastatic breast tumours for mutations in TC21 using a single-strand conformational polymorphism (SSCP)-based assay
Of nine primary and 15 metastatic breast tumour cDNAs analysed, none exhibited an altered pattem by SSCP
Summary
SSCPRNA was isolated using the RNAgents® Total RNA Isolation System (Promega). by a modification of the method of Wilkinson ( 1988) or using guanidinium isothiocyanate (Chirgwin et al 1979). cDNA was prepared as described previously (Barker et al 1995). CDNA was prepared as described previously (Barker et al 1995). Amplifications were carried out using cDNA derived from 0.2 jg of orginal RNA in 60 mM KCI 15 mm Tris-HCI pH 8.8. Each reaction was cycled 30 times at 94°C for 1 min, 550C for 2 min and 720C for 1 min. An aliquot of 4 of each reaction was mixed with 4 of SSCP gel loading buffer (95% deionized fonnamide, 0.025 M EDTA pH 8.0.0.005% bromophenol blue, 0.005% xylene cyanol). Undenatuned samples were kept on ice. An aliquot of 4 of eidter denatured or undenatured samples was loaded onto 6% acrylamnide, x TBE, 10% glycerol gels. Fragments were resolved by electophoresis at 5 W for 15 h; gels were dried and exposed to X-ray film at -70°C ovemnight
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