Gene expression profiling by DNA microarray in Colo741 cell line transfected by KRas-G12D or KRas-G12V

Mutations of the proto-oncogene KRas, together with the inactivation of the onco-suppressor genes APC and TP53, are considered to have an important role both in the carcinogenesis and in the colorectal tumor progression. The oncogene K-ras has activating mutations, especially in codon 12, in about 40% to 50% of colorectal carcinomas (Andreyev et al., 1998). The mechanism by which oncogenic K-ras alone contribuites to tumor progression in colon cancer is largely unknown. To this end, we have developed a model system where the colorectal adenocarcinoma cell line Colo741 has been stably trasfected with the Kras-G12V or Kras-G12D mutated oncogenes. Our results demonstrate how expression profiling data can be used to interpret changes in cellular characteristics induced by transfection and hence provide some clues as to the mechanisms by which different activated K-Ras impair processes mediated by endogenous wild-type KRas proteins. We made for the production of cell clones that could be utilized as useful in vitro models. The adenocarcinoma cell line Colo741 has been chosen to produce stable transfectants for two mutant forms of KRas2 (Gly12Val and Gly12Asp) and the wild-type coding sequence (as experiment control). After the transfection, the cells have been selected (with G418), cloned (by limited dilutions) and screened (by RT-PCR). The total RNAs, Dnase-treated, from five cell clones per condition, have been pooled. The samples, constituted by biotin-labeled cRNA, derived from a pool of five cell clones obtained from transfection of the adenocarcinoma cell line Colo741 with either pcDNA3.1-KRas2(Wt) or pcDNA3.1-KRas2(Gly12Val) or pcDNA3.1KRas2(Gly12Asp). They were hybridized to a Human Gene 1.0 ST array (Affymetrix). Two technical replicates have been performed for each condition.