Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA

Abstract

Trifluridine (FTD) and 2'-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

Figure 1

Structures of natural and analog pyrimidine nucleosides.

Figure 2

Comparison of dThd, FTD and FdUrd incorporation (A) into DNA and elimination of dThd and FTD (B) from DNA after a washout step. (A) HCT116 cells were treated for 1, 2, 4, 10 or 24 h with each compound at 1 μmol/l, and DNA incorporation was measured by LSC. (B) Following 24-h treatment with dThd or FTD, these compounds were washed out. At 24, 48 and 72 h following the washout step, the amount of dThd or FTD remaining incorporated into DNA was measured by liquid scintillation counting. Open circles, triangles and squares represent the incorporation of dThd, FTD and FdUrd, respectively.

Figure 3

Comparison of uptake levels of [³H]dThd (A), [³H]FTD (B), [³H]dUrd (C) and [³H]FdUrd (D) and the effects of nucleoside transporter inhibitors. The uptake of 1 μmol/l (1 μCi/ml) dThd, FTD, dUrd and FdUrd was measured for 1, 3, 5 and 10 min by LSC. HCT116 cells were pre-incubated for 10 min at 37°C in transport buffer (pH 7.4) before initiating the uptake assay. Closed circles, open triangles and squares represent cellular uptake with a control, 1 μmol/l NBMPR and 10 μmol/l DPM, respectively. Each result represents the mean ± SD (n=3).

Figure 4

Substrate specificity of DNA polymerase α for incorporating the triphosphate forms of FTD, dUrd and FdUrd into the complement DNA strand at sites matching adenine (A), guanine (B), thymine (C), or cytosine (D). Substrates were incubated for 30 min at 37°C in reaction buffer containing a single-stranded DNA oligonucleotide hybridized to an FITC-labeled primer (0.1 μmol/l) and DNA polymerase α. After reactions were completed, the samples were denatured and electrophoresed on a denaturing acrylamide gel at 300 V for 3.5 h.

Figure 5

Electron microscopy analysis of untreated HCT116 cells (A), or HCT116 cells treated with FTD (B) or FdUrd (C). Cells were treated in 60-mm culture dishes with 6 μmol/l FTD or 3 μmol/l FdUrd for 72 h, using a concentration found capable of 50% growth inhibition (data not shown) and subsequently fixed. After dehydration by ethanol treatment, samples were transferred to resin and polymerized. The acceleration voltage of an electron microscope was set at 80 kV, and observations were performed under ×714 magnification.

Figure 6

A potential mechanism of FTD and FdUrd incorporation into DNA via the dThd salvage pathway. In this model, both FTD and FdUrd are transported by both ENT1 and ENT2. The catalytic efficiency of TK1 for FTD phosphorylation is higher than that for FdUrd. FdUrd-triphosphate is degraded by DUT, while FTD-triphosphate is not a DUT substrate. Consequently, FTD incorporates into DNA effectively, compared with FdUrd.