Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling

Resistance to anticancer drugs such as the widely used antimetabolite 5-fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low-, intermediate- and high-resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM-2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium-binding protein family were suppressed. Acquisition of the intermediate-resistance phenotype included upregulation of the well-known drug resistance gene ABCC6 (ATP-binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time-depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high-resistance phenotype seems to be less demanding.