FTD was obtained from Yuki Gosei Kogyo Co., Ltd. (Tokyo, Japan). TPI was synthesized at Junsei Chemical Co., Ltd. (Tokyo, Japan). dThd, FdUrd and dUrd were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). [5-methyl-³H] dThd (25.0 Ci/mmol), [6-³H] dUrd (19 Ci/mmol), [6-³H] FdUrd (13.5 C i/mmol), and [6-³H] FTD (10.0 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA, USA). dNTPs were obtained from Takara Bio (Otsu, Japan). F₃dTTP was synthesized in house. Single-stranded DNA oligonucleotides were synthesized by Nihon Gene Research Laboratories (Sendai, Japan).

The human HCT116 colorectal cancer cell line derived from an adult male was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at 37°C with 5% CO₂ in McCoy’s 5A modified medium supplemented with 10% fetal bovine serum (FBS). Short tandem repeat profiling was performed to confirm the origin and authenticity of the HCT116 cell line.

HCT116 cells (1×10⁷) were seeded overnight in 175-cm² culture flasks. Subsequently, cells were incubated with drug mixtures containing tritium-labeled nucleosides at a final concentration of 1 μmol/l. The cells were harvested at 1, 2, 4, 10 and 24 h after treatment with each drug. DNA was extracted from cell pellets using a DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). The resulting DNA solutions were dissolved in 10 ml of Ultima Gold AB liquid scintillation fluid (Perkin-Elmer, Tokyo, Japan). Radioactivity in the samples was measured with a Tri-Carb 2900TR liquid scintillation analyzer (Perkin-Elmer), and the incorporation of tritium-labeled nucleosides into DNA was quantitated. DNA concentrations were determined using the Qubit^® dsDNA BR assay kit (Life Technologies, Carlsbad, CA, USA).

Nucleoside transport was evaluated by the silicone layer method (26). Briefly, cells were harvested the day before experiments began and suspended in transport medium containing 125 mmol/l NaCl, 4.8 mmol/l KCl, 5.6 mmol/l D-glucose, 1.2 mmol/l CaCl₂, 1.2 mmol/l KH₂PO₄, 12 mmol/l MgSO₄, and 25 mmol/l HEPES (pH 7.4). Cell suspensions were pre-incubated for 20 min at 37°C in transport medium, centrifuged, and the resultant cell pellets were resuspended in transport medium (pH 7.4) containing a radiolabeled nucleoside or nucleoside analog to initiate uptake. At appropriate times, 160-μl aliquots of cell suspension were withdrawn, and cells were separated by centrifugation through a layered mixture of silicone oil (SH550; Dow Corning Toray, Tokyo, Japan) and liquid paraffin (Wako Pure Chemicals Industries) with a density of 1.03 g/ml. Cell pellets were solubilized in 3 mol/l KOH and then neutralized with HCl. Next, cell-associated radioactivities were measured using a Tri-Carb 2900TR liquid scintillation analyzer. The cellular protein content was determined using the Qubit^® Protein assay kit (Life Technologies).

The pENTR221/TK1 and pENTR221/DUT constructs were obtained from Life Technologies. A Gateway LR reaction was performed to subclone the fusion partners into the Gateway bacterial expression vector pEXP1/DEST. Transformed BL21 Star^TM (DE3) pLysS cells were grown for 10 h at 37°C in LB medium containing 100 μg/ml ampicillin. Gene expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (0.1 μmol/l) that was cooled to 10°C. After 18 h of induction at 25°C, bacteria were collected by centrifugation, and pellets were stored at −135°C until purification. Bacterial pellets were resuspended in 10 ml BugBuster^® HT Protein Extraction reagent (Merck KGaA, Darmstadt, Germany) and incubated for 30 min at room temperature. Subsequently, the extract was clarified by centrifugal filtration with a 0.4-μm filter and purified with an AKTAprime plus with a HiTrap metal chelate column (GE Healthcare, Buckinghamshire, UK).

Ten micromolar tritium-labeled nucleosides and nucleoside analogs were incubated for 5 min at 37°C in 100-μl reactions containing 150 mmol/l Tris-HCl (pH 7.6), 6 mmol/l ATP, 15 mmol/l MgCl₂, 1% bovine serum albumin (BSA) and 20 ng recombinant human TK1. Reactions were stopped by the addition of 200 μl of methanol and clarified by ultrafiltration. After clarification, solutions were desiccated by exposure to N₂ gas, dissolved in 100 μl H₂O, and analyzed by radio-high-performance liquid chromatography (HPLC). HPLC was performed using a 150TR Flow System analyzer (Perkin-Elmer) and a Prominence Series HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with an ODS reverse-phase column (TSKgel ODS-100V, 250 mm × 4.6 mm, 3 μm; Tosoh Corp., Tokyo, Japan), with the eluent [10 mmol/l phosphate buffer (pH 3.0):Acetonitrile = 9:1] set to a flow rate 1.0 ml/min for 15 min. This procedure enabled quantitation of residual nucleosides and the production of nucleoside monophosphates formed in the reactions just described.

dUTPase assays were performed as previously described (27). Deoxynucleoside triphosphates (dUTP, dTTP, F₃dTTP and FdUTP; 30 mmol/l) were incubated for 30 min at 37°C in 100-μl reactions composed of 50 mmol/l Tris-HCl (pH 7.4), 4 mmol/l MgCl₂, 2 mmol/l 2-mercaptoethanol, 0.1% BSA and 100 ng human recombinant DUT. Reactions were stopped by the addition of 11 μl 4.2 mol/l perchloric acid per reaction. Samples were clarified by centrifugation for 10 min at 13,000 rpm. Supernatants (70 μl) were neutralized with 32 μl of 1 mol/l K₂HPO₄, followed by a 10-min centrifugation at 13,000 rpm. Supernatants (10 μl) were resolved by HPLC as described above to quantify residual dUTP and newly formed dUMP.

Recombinant DNA polymerase α (CHIMERx, WI) activity was assayed according to the manufacturer’s recommended protocol. The substrates (dNTPs) were incubated for 30 min at 37°C with 60 mmol/l Tris-HCl (pH 7.0), 5 mmol/l MgCl₂, 0.3 mg/ml BSA, 1 mmol/l DTT, 0.1 μmol/l, 0.5 U DNA polymerase α, fluorescein isothiocyanate (FITC)-labeled primer (5′-FITCGTAAAACG ACGCC AGT-3′), and 0.1 μmol/l synthetic DNA template (5′-TCG GACTGGCCG TCG TTTTAC-3′, 5′-TCG CACTGGCCG TCG TTTTAC-3′, 5′-TCG AACTGGC CG TCG TTTTAC-3′, or 5′-TCG TACTGGCCGTCGTTTT AC-3′). The underlined sequences represent nucleotides that were modified partially to confirm the sites where F₃dTTP and FdUTP were inserted.

After the enzyme reactions were complete, samples were resolved by electrophoresis on an ERICA-S system (DRC, Tokyo, Japan) at 300 V for 3.5 h, and FITC-fluorescence was detected using an ImageQuant LAS 4010 imager (GE Healthcare).

For electron microscopy studies, samples were fixed with 2% paraformaldehyde and 2% glutaraldehyde, after which they were exposed to 2% osmium tetroxide. Following dehydration with a graded ethanol series, samples were transferred to resin quetol 812 (Nisshin EM, Tokyo, Japan) and polymerized at 60°C for 48 h. The blocks were sectioned at 70 nm with an Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany), and sections were placed on copper grids and stained with 2% uranyl acetate and lead stain solution (Sigma-Aldrich, Carlsbad, CA, USA). The grids were evaluated using a JEM-1200EX transmission electron microscope (JEOL, Tokyo, Japan).

Initially, we compared the rates of FTD and FdUrd accumulation into DNA in HCT116 cells with that of the native substrate dThd, using final nucleoside concentrations of 1 μmol/l. Fig. 2A shows the time course for nucleoside accumulation at 1, 2, 4, 10 and 24 h post-exposure. Tritium-labeled nucleosides were used as substrates and were quantified by liquid scintillation counting (LSC). dThd and FTD were incorporated into DNA at comparable levels at the 1- and 2-h time-points. dThd incorporation increased linearly until 10 h, while saturation of FTD incorporation occurred by 4 h, reaching approximately half that observed with dThd (dThd, 41.5 pmol/μg DNA; FTD, 17.6 pmol/μg DNA). However, the level of FdUrd incorporation into DNA was relatively low. For example, after a 24-h incubation, FdUrd incorporation was one sixteenth of that observed with FTD (FdUrd, 1.1 pmol/μg DNA), which is close to the limit of detection.

Next, we compared the susceptibility of FTD to elimination from DNA with that of dThd. In this assay, cells were treated with 1 μmol/l dThd and FTD for 24 h, after which drug-free medium was added and the cells were incubated for an additional 24, 48 or 72 h. Fig. 2B shows the residual ratios of dThd to FTD at 24, 48 and 72 h after the washout steps. Both dThd and FTD incorporation into DNA decreased gradually after the washout step, and only minor differences were observed between dThd and FTD (dThd 34.1%, FTD: 29.0%). We also attempted to assess FdUrd elimination from DNA after the washout step; however, it was nearly undetectable in this assay and could not be evaluated.

To monitor the transport of FTD and FdUrd into the cytoplasm and their intracellular uptake, the inhibitory effects of the nucleoside transporter inhibitors 6-[(4-nitrobenzyl) thio]-9-(β-D-ribofuranosyl) purine (NBMPR) and dipyridamole (DPM) were analyzed and compared to the native substrates dThd and dUrd, respectively (Fig. 3). The concentration of inhibitors used was based on the concentration of NBMPR required to inhibit ENT1, or that required for DPM inhibition of both ENT1 and ENT2 (28).

After incubating HCT116 cells with dThd or FTD for 10 min, we found that the inhibitory effect of 1 μmol/l NBMPR was less than that of 10 μmol/l DPM. For instance, the levels of cellular dThd uptake at 10-min post-treatment with a vehicle control, 1 μmol/l NBMPR, or 10 μmol/l DPM were 1.59±0.06, 0.47±0.06 or 0.04±0.01 nmol/mg protein, respectively, while those for FTD uptake were 1.00±0.04, 0.07±0.01 and 0.03±0.02 nmol/mg protein, respectively (Fig. 3A and B).

HCT116 cells were also incubated with dUrd and FdUrd for 10 min, and inhibition by 1 μmol/l NBMPR was again lower than that by 10 μmol/l DPM. The levels of dUrd uptake at 10-min post-incubation with a vehicle control, 1 μmol/l NBMPR, or 10 μmol/l DPM were 1.80±0.08, 0.58±0.09 and 0.05±0.01 nmol/mg protein, respectively, and those of FdUrd were 4.90±0.27, 0.29±0.07 and 0.03±0.00 nmol/mg protein, respectively (Fig. 3C and D). These results indicate that all nucleosides tested in these experiments could be recognized and transported into cells by both ENT1 and ENT2.

To evaluate the substrate specificity of FTD, dUrd and FdUrd for TK1, recombinant human TK1 was incubated with tritium-labeled substrates. The K_m values determined for each tested nucleoside revealed that dThd analogs had a higher affinity for TK1 than for dUrd analogs (Table I). The V_max values of dThd-based compounds exceeded those of the dUrd-based compounds. FTD and FdUrd showed higher affinities compared to the native form of these nucleosides, namely, dThd and dUrd.

To evaluate the catalytic efficiency of TK1 in phosphorylating dThd, FTD, dUrd and FdUrd, k_cat/K_m ratios were calculated. The k_cat/K_m values of dThd and its analog FTDs tended to be higher than those of dUrd and its analog FdUrd (Table I). TK1 showed higher catalytic efficiency in phosphorylating FTD than it did with dThd, and the catalytic efficiency of FTD phosphorylation by TK1 was ~4-fold higher than that of FdUrd.

Pyrimidine triphosphate incorporation into DNA is strictly regulated by DUT (29,30) and high DUT expression is reported in various types of cancer, suggesting that DUT may confer resistance to 5-fluoropyrimidines (24,30). To determine whether FTD is substrate of DUT, we performed enzymatic assays with recombinant human DUT and either FdUTP or F₃dTTP (Table II). Although DUT efficiently converted dUTP and FdUTP to their monophosphate forms (dUMP and FdUMP, respectively), dTTP and F₃dTTP were not found to be substrates for DUT.

Next, we investigated whether F₃dTTP and FdUTP serve as substrates for DNA polymerase α, the responsible enzyme for catalyzing DNA replication. We analyzed extension reactions by DNA polymerase α using dNTPs (as control substrates) and the triphosphate forms of dUrd, FTD and FdUrd, and a synthetic single-stranded DNA template (Fig. 4). F₃dTTP and FdUTP, dThd and dUrd were incorporated into the nascent strand at sites matching adenine on the template strand, but not at guanine, cytosine or thymine sites.

Finally, to compare DNA incorporation data with observable phenotypes, we performed morphological analyses of HCT116 cells treated with FTD and FdUrd by electron microscopy (Fig. 5). Swollen nuclei and structural abnormalities of nucleoli were observed in both FTD- and FdUrd-treated cells (relative to untreated control cells), with the degree of alteration caused by FTD exposure much stronger than that caused by FdUrd. Furthermore, the perinuclear heterochromatin content was decreased by both FTD and FdUrd treatment.