| Summary: | <p>Abstract</p> <p>Background</p> <p>Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type <it>TP53 </it>5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure.</p> <p>Results</p> <p>5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (<it>ATF3</it>, <it>GADD34</it>, <it>GADD45A</it>, <it>PCNA</it>), cell cycle-regulatory (<it>CDKN1A</it>), and apoptosis-regulatory pathways (<it>FAS</it>), and apoptosis induction in the parental and resistant cell lines. Levels of 5-FU incorporation into DNA were similar for the cell lines. The pattern of cell cycle progression during recovery demonstrated consistently that the 5-FU-resistant cell lines had the smallest S phase fractions and the largest G<sub>2</sub>(/M) fractions. The strongly 5-FU-resistant ContinD cell line had the smallest S phase arrests, the lowest CDKN1A levels, and the lowest levels of 5-FU-induced apoptosis throughout the treatment and recovery periods, and the fastest recovery of exponential growth (10 days) compared to the other two cell lines. The moderately 5-FU-resistant ContinB cell line had comparatively lower apoptotic levels than the parental cells during treatment and recovery periods and a recovery time of 22 days. Mitotic activity ceased in response to drug treatment for all cell lines, consistent with down-regulation of mitosis-regulatory genes. Differential expression in response to 5-FU treatment was demonstrated for genes involved in regulation of nucleotide binding/metabolism (<it>ATAD2</it>, <it>GNL2</it>, <it>GNL3</it>, <it>MATR3</it>), amino acid metabolism (<it>AHCY</it>, <it>GSS</it>, <it>IVD</it>, <it>OAT</it>), cytoskeleton organization (<it>KRT7</it>, <it>KRT8</it>, <it>KRT19</it>, <it>MAST1</it>), transport (<it>MTCH1</it>, <it>NCBP1</it>, <it>SNAPAP</it>, <it>VPS52</it>), and oxygen metabolism (<it>COX5A</it>, <it>COX7C</it>).</p> <p>Conclusion</p> <p>Our gene expression data suggest that altered regulation of nucleotide metabolism, amino acid metabolism, cytoskeleton organization, transport, and oxygen metabolism may underlie the differential resistance to 5-FU seen in these cell lines. The contributory roles to 5-FU resistance of some of the affected genes on these pathways will be assessed in future studies.</p>
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