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Ras proteins are plasma-membrane bound proteins regulated by intrinsic GTPase activity. Mutations in ras proteins result in the constitutive protein activation and stimulation of downstream ras pathways, reactive oxygen species production, and DNA damage. Alternative splicing of the fourth exon in K-ras mRNA results in splice variants, the longer 4A form which is similar to H-ras and N-ras in having C-terminal S-palmitoylation as part of cell membrane targeting, and the 4B variant which is unique in presenting a polylysine C-terminal tail. While the K-ras 4B is widely expressed and the most studied, the K-ras 4A variant has distinct localization and functional properties. The potential specific roles of these two variants in tumorigenesis are poorly understood. The levels of splice variants were quantified in human lung adenocarcinoma cell lines with codon 12 K-ras mutations (A549 and H441), as compared with a nontransformed human lung line (HPL1D). Variant-specific reverse transcription and real-time polymerase chain reaction detected that K-ras 4A mRNA was significantly upregulated in A549 (1.7-fold, p<0.005; 4B variant: p=0.34; total: p=0.87) and H441 (4.8-fold, p<0.001; 4B: 1.7-fold, p<0.05; total: 1.8-fold, p<0.03). Results were not significantly different for proliferating vs post-confluent cell cultures. Most strikingly, the level of the K-ras 4A mRNA was concordant with the amount of superoxide (1.6-fold increase in A549 and 7-fold elevation in H441), measured by nitroblue tetrazolium, suggesting a link between the 4A variant specifically and reactive oxygen species. Mutant and wild-type K-ras transcripts were also quantified by pyrosequencing. This confirmed only mutant alleles in A549 cells, and revealed that mutant and wild-type K-ras alleles were equally expressed in H441 cells. In sum, these findings provide the first evidence that (1) the K-ras 4A variant is differentially upregulated in some lung cancer cells, (2) mRNA for mutant K-ras variants is not favored over wild-type by upregulation, and (3) increased reactive oxygen species in lung cancer cells may be specifically related to level of K-ras 4A, and this could be fundamentally important to the role of K-ras in the malignant phenotype.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA