N-ras protein: frequent quantitative and qualitative changes occur in human colorectal carcinomas

Point mutation and overexpression are recognized mechanisms for ras activation in malignancy. However, little information is available on overexpression of N-ras protein compared with H- or K-ras proteins, as N-ras-specific antibodies have only recently become available. For comparative analyses of ras protein levels, we have probed Western blots of extracts from 9 normal human tissues and 55 pairs of colorectal carcinoma and matched control mucosa, using monoclonal antibodies (MAbs) specific for H-, K- or N-ras proteins. On multi-tissue blots, N-ras protein was more highly expressed in colon than in the other human tissues analyzed, suggesting a role for N-ras in colorectal function. N-ras protein levels in multiple independent extracts of normal colon mucosa were consistently higher than either K-ras protein or H-ras protein levels. In 69% of colon carcinomas, N-ras protein levels were increased an average of 4.8-fold over normal mucosa. Overexpression of K-ras protein was also observed in colon cancers but less frequently (13% of cases) than N-ras protein. H-ras protein levels were too low for comparative studies. Alterations in N-ras protein mobility, possibly reflecting increased post-translational processing, were also detected in 42% of colon carcinomas. N-ras protein, typically present as a single 23 kDa band in normal mucosa, was expressed in some cancers as a 22 kDa band or as multiple bands of 20-23 kDa. Sequencing of N-ras DNA from 6 carcinomas with these variations in protein mobility did not reveal mutations in codons 12, 13, 59 or 61. Thus, frequent quantitative and qualitative changes in N-ras protein expression, which do not appear to correlate with the presence of typical N-ras point mutations, result in abnormal N-ras protein patterns in human colorectal carcinomas.