Figure 1 - Mutant K-Ras, but not mutant N-Ras, promotes hyperplastic growth in the colonic epithelium.

(a) Expression analysis of Kras and Nras in the colonic epithelium. We used Mcm6, which is expressed only in undifferentiated cells at the bottom of the crypt, as a control. Kras is more highly expressed at the top of the crypt, whereas Nras is uniformly expressed throughout the crypt. Expression of Mcm6, Kras and Nras was normalized to the expression of the TATA-box binding protein (Tbp) gene. (b) Biochemical detection of activated K-Ras and N-Ras. Activated (GTP-bound) forms of each protein can be detected in lysates from colonic epithelium, but only in mice carrying the conditional (LSL-G12D) allele and expressing Cre recombinase from the Fabpl promoter. (c) Hematoxylin and eosin staining of normal colonic epithelium. Yellow shading denotes the normal proliferative zone, and green shading marks the zone of differentiation. All animals were killed between 4–6 months of age. (d) Hematoxylin and eosin staining of colonic epithelium expressing activated K-Ras^G12D showing crypt hyperplasia. (e) Hematoxylin and eosin staining of colonic epithelium expressing activated N-Ras^G12D. (f) Signaling downstream of K-Ras^G12D and N-Ras^G12Din vivo. In quantitative protein blots of whole tissue lysates (see example in left), mutant K-Ras activates Mek and Erk. Mutant N-Ras, by contrast, does not activate Mek or Erk. Both K-Ras^G12D and N-Ras^G12D downregulate phospho-Akt, whereas neither seems to significantly affect phospho-Jnk. ^*P < 0.05, Wilcoxon rank sum test. The activation state of each molecule is measured as the ratio of phospho-protein to total protein. (g,h) Immunohistochemical detection of phospho-Mek. Few cells in the normal colonic epithelium (g) express phospho-Mek, whereas all cells expressing mutant K-Ras stain positively (h). (i,j) Immunohistochemical detection of phospho-Erk. Phospho-Erk is readily detectable in the differentiated cells at the top of the colonic crypt expressing K-Ras^G12D (j), but not in the proliferative cells at the bottom of the crypt. In h and j, the height of the proliferative progenitor zone is represented by the yellow line. Scale bar in all panels, 50 m. Error bars, s.d.