Analysis of expressed N- ras mutations in human melanoma short-term cell lines with allele specific restriction analysis induced by the polymerase chain reaction

Abstract

Mutations in ras genes have been found in the DNA of numerous cancer types including melanomas, but the expression of these mutations in melanomas has not yet been addressed. We have used the polymerase chain reaction (PCR) and allele-specific restriction analysis (ASRA) to determine the frequency of expressed N- ras mutations on 25 short-term melanoma tissue culture samples. N- ras cDNA generated using reverse transcriptase from whole cells was used as the PCR template. 14 secondary melanoma cultures that varied in differentiation patterns were analysed. Only 2 were found to express N- ras mutations; in both, the mutation was localised to one of the first two positions of the 61st codon of N- ras. These tumour lines, KMI-M8412a and KMI-M8412b, were established from separate tumour deposits in the same patient. Codons 12 and 13 were found to be free of mutations in all of the lines studied. 8 primary melanomas and 3 unclassified skin lesions were also analysed and found free of N- ras mutations. These results suggest that N- ras may not play such an important role in melanoma tumorigenesis as is speculated by others.