Allele-specific detection of K-ras oncogene expression in human non-small-cell lung carcinomas.
Point mutations in codon 12 of the K-ras oncogene are frequent in human lung adenocarcinomas. To study the expression of the K-ras gene in these tumors we have developed a mRNA detection technique based on the polymerase chain reaction (PCR). By this technique, K-ras expression can be detected semi-quantitatively in samples of less than 100 ng total RNA. Hybridization of the amplified cDNA sequences with mutation-specific oligonucleotides allows separate quantification of the expression of normal and point-mutated alleles in a single sample. RNA samples from 24 human non-small-cell lung carcinomas (NSCLC), from 2 lung metastases of colonic adenocarcinomas, from 3 human lung adenocarcinoma cell lines, and from normal lung tissue were analyzed. In most tumors, expression of K-ras was detected at levels equal to or several times higher than those found in normal lung tissue. A lung metastasis from a colon adenocarcinoma, known to contain an amplified K-ras gene, highly over-expressed the K-ras gene. In those tumors in which the K-ras oncogene was activated by a point mutation, both alleles of the gene were expressed. Our results show that a high over-expression of K-ras is a rare event in human lung carcinomas, but that a certain degree of over-expression of the mutated allele can be demonstrated in tumors with an activated K-ras gene. With the technique we describe here, adequate estimation of the expression of specific genes in minimal amounts of tumor cells becomes possible.
Slebos
R J
RJ
Department of Experimental Therapy, The Netherlands Cancer Institute, Amsterdam.
Habets
G G
GG
Evers
S G
SG
Mooi
W J
WJ
Rodenhuis
S
S
D000230:Adenocarcinoma / Q000235:genetics; D000483:Alleles; D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D002460:Cell Line; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D011905:Genes, ras; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification
D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D012334:RNA, Neoplasm