As an example, we detail the detection of a point mutation at codon 61 of the human N-ras gene in DNA from a formalin-fixed paraffin-embedded malignant melanoma specimen.
Mutational analysis of human NRAS genes in malignant melanoma: rapid methods for oligonucleotide hybridization and manual and automated direct sequencing of products generated by the polymerase chain reaction.
Each example of an activated ras gene showed a mutation at the 61st codon of the protein, with the exception of one melanoma which showed a mutation at codon 13 of the N-ras gene; (2) all the melanomas displaying an activated ras gene had a similar cell surface phenotype and appear to come from a similar phase of differentiation; (3) 5-6% of noncultured primary and metastatic melanomas have mutated ras genes; (4) no ras gene mutations were found in any precursor lesion, specifically normal nevi and dysplastic nevi; (5) immunoperoxidase analysis of paraffin-embedded specimens indicated no quantitative or qualitative alterations in p21 expression that correlate with tumor progression; (6) there were no observable differences in p21 expression between melanoma cells growing exponentially or in plateau phase, or between melanoma cells with or without ras mutations; nor were any cell kinetic differences found between cells with and without mutated ras genes.
However, the analysis of the same DNAs for a different polymorphism based on the presence of additional TaqI sites in the variable tandem repeat region of HRAS1 showed that the total frequency of a group of allelic variants, named Tp, was significantly higher in melanoma patients than in normal donors.
To evaluate whether N-RAS could directly modulate LAK susceptibility to lysis, N-RAS/61+ gene was transfected in two N-RAS wild type (N-RAS/61-) 665/2 melanoma clones by a cosmid vector.
Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein.
Tumors grown in SCID mice injected with human melanoma carrying activated N-ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants.
We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced.
The increase in Bcl-2 expression mediated by mutated ras therefore qualifies as a rational explanation for the enhanced chemoresistance of human melanoma expressing mutated N-Ras.
We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing.
Mutations in the ras genes are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in cutaneous melanoma.
Novel findings with respect to melanoma include a cell line possessing a 2 base-pair substitution in BRAF exon 11 and a case harboring mutations in both BRAF exon 11 and N-RAS exon 3.
We analyzed exons 1, 11, and 15 of the BRAF gene and exons 1 and 2 of the N-ras gene for mutations in 38 metastatic melanomas by PCR-single-strand conformation polymorphism and direct sequencing.
We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique.
Previously, we analyzed a large series of paired primary and metastatic melanomas for NRAS codon 61 mutations and showed that they arise early and are preserved during tumor progression.