Molecular characterization of established human colorectal carcinoma cell lines (HCT-8) made resistant to 5-fluorouracil (5-FU) by different selective schedules

5-FU and 5-fluorodeoxyuridine (5-FdUrd) are still the two main drugs for treatment of advanced colorectal cancer. The main mechanisms of action of 5-FU are represented by thymidylate synthase (TS) inhibition and incorporation of antimetabolite into RNA. The first mechanism prevails for long-term exposure to 5-FU, the second for short-term exposure to 5-FU. On this basis, 5-FU may act in two distinct molecular manners in the clinic, depending on the administration schedule. Both regimens, however, inevitably lead to 5-FU resistance. To provide some insight into molecular mechanisms of 5-FU clinical resistance in colorectal cancer, we hypothesised that different in vitro exposure schedules of human colorectal cancer cell lines mimicking clinical infusional or bolus regimens could lead to differential gene expression. Resistant HCT-8 colorectal cancer cell lines, namely HCT-8/FUI/15R and HCT8/FUB/2R, were selected from parental sensitive HCT-8 cells by long-term and short-term exposure schedules, respectively. In all cell lines, 437 human genes, including 32 cancer genes and 9 housekeeping genes, were analysed by Atlas select cDNA Expression Human Tumour Array (Clontech, CA). PolyA+ RNAs were isolated, DNAse digested, retrotranscribed, radiolabelled and used as cDNA probes. Cell death morphology and cytokinetic parameters were analysed. Inhibitory effects of 5-FU and other anticancer drugs on cell growth (by sulforhodamine B assay) and baseline expression of genes involved in the sensibility/resistance to 5-FU (by RT-PCR) were evaluated. Expression levels of the 437 tested genes were not substantially different between HCT8/FUB/2R and HCT-8 cell lines. Sixty-three genes were differentially expressed in HCT8/FUI/15R cells compared to the parental cell line: 13 genes, including 3 chemoresistance-related genes, were selectively overexpressed, whilst 3 genes were switched-off. These cell lines were also substantially different for morphology of cell death. HCT8/FUB/2R cells were substantially more resistant to 5-FU in comparison to HCT8/FUI/15R cells after 4 and 72 h exposures. No substantial differences were observed among resistant and parental cells in sensitivity to SN-38 and oxaliplatin. Expression of TS, thymidine phosphorylase, bcl-2 and ERCC1 genes was comparable, whereas expression of the dihydropyrimidine dehydrogenase gene was markedly increased in both resistant cell lines. These results support the importance of the 5-FU schedule selected to treat advanced colorectal cancer. Analysis of in vitro response to different schedules of anticancer drugs, either alone or in combination, utilizing the cell line models described herein may help in designing clinically useful drug regimens.