Specific K-ras2 mutations in human sporadic colorectal adenomas are associated with DNA near-diploid aneuploidy and inhibition of proliferation

Abstract

Recent studies indicate that p21ras proteins mediate their multiple cell functions through interactions with multiple effectors and that the number of new effectors is growing. We recently reported that K-ras2 mutations in human colorectal adenomas were associated with chromosome instability and proliferation changes. In the present study, we extend these previous observations. Hereditary and multiple (n > or = 5) adenomas and adenomas with early cancer were excluded. Dysplasia was moderate in 91 cases and high in 25, and the median adenoma size was 1.5 cm. K-ras2 spectrum analysis was done by sequence-specific oligonucleotide hybridization using nuclear suspensions provided by analysis and sorting of multiparameter flow cytometry. In particular, tissue inflammatory cells were separated for DNA diploid tumors, whereas DNA aneuploid epithelial subclones were analyzed separately. K-ras2 mutations and DNA aneuploidy were both detected in 29 of 116 (25%) cases. DNA aneuploid index was in the near-diploid region in the majority of cases. DNA aneuploidy was strongly associated with G-->C/T transversions. An association was also found between low S-phase values and G-->A transitions. These findings were confirmed using multivariate logistic regression analysis to account for the effects of size, dysplasia, site, type, age, and sex. These data suggest that specific K-ras2 mutations in a subgroup of human sporadic colorectal adenomas play a role in chromosome instability and, contrary to expectations, are associated with inhibition of proliferation.

Figure 1.

Examples of multiparameter FCM of DAPI-stained nuclear suspensions obtained from dysplastic areas of human sporadic colorectal adenomas. Top: Forward and side scatter parameters were used to gate out inflammatory cells (corresponding to A2 and B2 subregions) and correct S-phase fraction values among DNA diploid cases. Bottom: Detection of a DNA aneuploid near-diploid peak overlapping with the DNA peak of diploid inflammatory cells. The FCM sorting of an enriched epithelial cell component was done to improve the sensitivity of PCR and sequence-specific oligonucleotide analysis of the K-ras2 spectrum.

Figure 2.

Top: DI aneuploid values (DI ≠ 1) detected by multiparameter FCM. Center: S-phase fraction values, respectively, below and above the median value of 7.3%. S-phase values in DNA diploid cases were corrected by subtracting the inflammatory cell component. Bottom: S-phase values versus all DIs.

Figure 3.

Examples of K-ras2 mutation spectrum analysis. K-ras2 mutation analysis was for wild type in codons 12 and 13 (wild-type codon 12 GGT-gly and codon 13 GGC-gly), six possible mutations in codon 12 (AGT, TGT, CGT, GAT, GCT, and GTT), and two mutations in codon 13 (AGC and GAC). K-ras2 wild-type controls were the following: peripheral blood lymphocytes of healthy donors (g6), SW837 cell line with heterozygous TGT mutation (h2), NIH3T3 cell line with transfected CGT human mutated K-ras2 (h3), SW480 cell line with homozygous GTT mutation (h4), and DLD-1 cell line with heterozygous GAC mutation (h5). All other spots correspond to different wild-type or mutated adenoma samples. Of particular interest are adenomas with TGT mutation in both diploid and aneuploid subclones (e2 and e3) and adenomas with GTT (a6 and b1) and GAT (c2 and c3) mutations in both low-grade and high-grade dysplasia components.