Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery

Abstract

Background: 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 TP53 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.

Results: 5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (ATF3, GADD34, GADD45A, PCNA), cell cycle-regulatory (CDKN1A), and apoptosis-regulatory pathways (FAS), 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 G2(/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 (ATAD2, GNL2, GNL3, MATR3), amino acid metabolism (AHCY, GSS, IVD, OAT), cytoskeleton organization (KRT7, KRT8, KRT19, MAST1), transport (MTCH1, NCBP1, SNAPAP, VPS52), and oxygen metabolism (COX5A, COX7C).

Conclusion: 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.

Figure 1

Cell counts during recovery periods following drug removal: cell counts were measured throughout the respective recovery periods for each cell line following a shift to drug-free medium at 24 hours (Day 1). The dashed (---) line shows the number of viable cells in untreated exponentially-growing cultures.

Figure 2

Cell cycle distributions during recovery periods following drug removal: fractions of cells in (a)G₁, (b)S, and (c)G₂ cell cycle phases were measured at intervals during the respective recovery periods for each cell line.

Figure 3

Apoptotic fractions during recovery periods following drug removal: apoptosis induction in 5-FU-treated HCT116 cell lines during the respective recovery periods for each cell line. The dashed line shows the levels of natural (spontaneous) apoptosis in untreated control cells.

Figure 4

Incorporation of 5-FU into DNA of parental and 5-FU-resistant HCT116 cell lines in response to 8 and 24-hr. 5-FU treatment: Incorporation of 5-FU into DNA is given as the ratio of disintegrations per minute (dpm) per μg DNA. The dashed line represents background dpm levels.

Figure 5

Cell growth in parental and 5-FU-resistant HCT116 cell lines in response to 5-FU treatment for 24 hours: cell growth at 8 and 24 hours after addition of 5-FU to the media in (a) parental cells, (b) ContinB cells, (c) ContinD cells.

Figure 6

Mitotic fractions in parental and 5-FU-resistant HCT116 cell lines in response to 5-FU treatment for 24 hours: mitotic fractions in all cell lines at 8 and 24 hours after 5-FU addition, showing gradual cessation of mitosis over the 24-hour treatment period.

Figure 7

Cell cycle progression in parental and 5-FU-resistant HCT116 cell lines in response to 5-FU treatment for 24 hours: (a) cell cycle distributions showing G₁/S boundary arrests at 8 hours and S phase arrests at 24 hours, showing S phase arrests at 24 hours; (b)-(d) G₁, S and G₂M fractions in all cell lines at 8 and 24 hours after 5-FU addition.

Figure 8

DNA damage response and cell cycle-regulatory protein and transcript levels in 5-FU-treated parental and resistant HCT116 cell lines: (a) p53 protein and TP53 transcript levels at 8 and 24 hours in 5-FU-treated HCT116 cell lines and their corresponding untreated controls; (b) GADD45A protein and transcript levels at 8 and 24 hours in 5-FU-treated HCT116 cell lines and their corresponding untreated controls; (c) CDKN1A protein and transcript levels at 8 and 24 hours in 5-FU-treated HCT116 cell lines and corresponding untreated controls; (d) MYC protein and transcript levels at 8 and 24 hours in 5-FU-treated HCT116 cell lines and corresponding untreated controls. For the bar charts that present gene expression levels, colorless bars depict the parental cell line, black bars the ContinB cell line, and vertically-striped bars the ContinD cell line.