HrasKI homozygous mice developed significantly more lung tumours than WT littermates. The increase in lung tumour number correlated with the number of HrasKI alleles. Error bars indicate standard deviations, and statistics were performed using the Wilcoxon Mann-Whitney test.

Kras-4A staining was observed in a sub-population of Clara cells in WT129/Sv (a) and FVB/N mice (b) but absent in homozygous KrasKI mice (expressing only Kras-4B) (c). The staining was observed throughout the bronchial tree, terminating at bronchio-alveolar duct junctions, locations of the BASCs. In contrast, papillary adenomas from 129/Sv mice do not show significant staining for Kras4A (d), although adjacent bronchial structure showed positive staining (d, inset).

Analysis of Ras protein levels in lungs of untreated mice showed elevated levels of Hras protein and complete absence of Kras protein in HrasKI homozygous mice. Mice homozygous and heterozygous for the KrasKI allele express similar Kras protein levels to WT mice. No apparent differences in activation of Akt and Erk, as measured by protein phosphorylation, and in level of p38α were observed among mice of the indicated genotypes.

(a) KrasKI homozygous mice were almost free of lung tumours, while KrasKI heterozygous mice remained susceptible to lung carcinogenesis, and developed significantly more lung tumours than WT mice. Mutations in these tumours were found exclusively in the endogenous Kras gene and not the KrasKI allele. (b) Kras^LA2 mice harboring the KrasKI allele, which generates only the 4B isoform, developed almost 3-fold more lung tumours than those carrying an intact Kras gene capable of generating both 4A and 4B isoforms. The HrasKI allele in Kras^LA2 displayed an inhibitory effect on tumor number similar to WT mice. Error bars indicate standard deviations, and statistics were performed using the Wilcoxon Mann-Whitney test.

WT and HrasKI mice developed a spectrum of papillary (a), mixed (b) and solid adenomas (c). Whereas WT animals predominantly developed papillary adenomas with occasional mixed and solid tumours, the HrasKI mice tended to develop more mixed and solid adenomas, sometimes with prominent intrabronchiolar extension (c, inset, marked by asterisk). The dashed line in (b) marks the boundary between the papillary (left) and solid (right) portions of the mixed adenoma. Several solid adenomas from HrasKI mice contained epithelioid cells (d, inset enlarged in e) which stained positive for (f) cytokeratin 8/18 and negative for PAS (g). Occasional macrophage clusters (f, keratin-negative cells) were also present in the epithelioid adenomas. Goblet cell metaplasia served as a positive control for PAS/mucin (g, inset). These adenomas displayed features of alveolar type II pneumocytes like their papillary counterparts (not shown) as they stain positively for SPC (h) and negatively for CCA/CC10 (i). The adjacent bronchiolar epithelium stained positive for CCA/CC10 as expected (i, inset).