Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA

Abstract

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.

Figure 1

Pathways of 5-FU metabolism and DNA toxicity. Metabolites are shown in bold and enzymes in italics: 5-FU, 5-fluorouracil; 5-FdUrd, 5-fluorodeoxyuridine; 5-FdUMP, 5-fluorodeoxyuridine monophosphate; 5-F-dUTP, 5-fluorodeoxyuridine triphosphate; dUTP, deoxyuridine triphosphate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; dTTP, deoxythymidine triphosphate; TP, thymidine phosphorylase; NMK, nucleotide monophosphate kinase; NDK, nucleotide diphosphate kinase; TK, thymidine kinase; dUTPase, dUTP nucleotidohydrolase; TS, thymidylate synthase; DPDH, dihydropyrimidine dehydrogenase.

Figure 2

Measurement of [dUTP + 5-F-dUTP] and TTP levels in HT29, MEF, HeLa and DT40 cell extracts in the presence and absence of 5-FdUrd (7.5 µM) and RTX (0.5 µM). (A) Denaturing polyacrylamide gel analysis of template extension reaction using NTP extracts obtained from HT29 cells cultured in the absence and presence of 5-FdUrd using low folate RPMI media with dialyzed FBS. T, DNA template primer. The minor n - 1 band in each lane arises from pyrophosphorolysis of the template. (B), (C), (D) TTP and [dUTP + 5-F-dUTP] levels (pmol/10⁶ cells) measured in methanol extracts obtained from HT29, HeLa or MEF cells grown in the absence or presence of 5-FdUrd or RTX using standard or low folate (LF) RPMI or DMEM media. (E, F) TTP and [dUTP + 5-F-dUTP] levels (pmol/10⁶ cells) measured in methanol extracts obtained from DT40 cells grown in the absence or presence of 5-FdUrd or RTX using standard or low folate (LF) chicken media (CM).

Figure 3

Toxicity of 5-FdUrd and RTX to DT40 cell lines that express or are deficient in UNG activity. The IC₅₀ values for 5-FdUrd are 6 ± 1 nM (aid^−/− ung^−/−) and 8 ± 1 nM (aid^−/−) and for RTX 9 ± 2 nM (aid^−/− ung^−/−) and 8 ± 2 nM (aid^−/−). The error bars are standard errors from three replicate determinations.

Figure 4

Representative single-turnover and steady-state measurements of glycosylase activity. (A) Single-turnover kinetic time courses for hSMUG1 catalyzed excision of 5-Fu from the F/A duplex as a function of enzyme concentration. (B) hSMUG1 concentration dependence of the observed rate constants. The kinetic parameters are reported in Table 2. (C) Steady-state kinetics for UNG2 excision of 5-FU from the F/G duplex (Table 1). Upper and lower bands in gel correspond to substrate and product bands, respectively.

Figure 5

In vitro kinetic excision parameters of purified hSMUG1 and hTDG relative to hUNG2 for an array of DNA substrates. (A) k_cat of hSMUG1 and hTDG relative to hUNG2. (B) k_cat/K_m of hSMUG1 and hTDG relative to hUNG2. Kinetic parameters for hTDG were previously reported (14, 15) under identical buffer and substrate conditions.

Figure 6

Excision of various DNA lesions by nuclear extracts obtained from cells grown in the absence (black bars) and presence (red bars) of 1 µM 5-FU for 48 h. (A) Representative gel of various DNA substrates incubated with MEF nuclear extracts in the presence and absence of the specific inhibitor of UNG (UGI). Top and bottom bands correspond to substrate (S) and product (P) bands, respectively. Identity of the central base pair of the substrate is listed above each lane (−/+ indicates the absence or presence of UGI). The two product bands are due to fractional removal of the terminal deoxyribose sugar during hot alkali processing of abasic sites. (B, C, D) Percent cleavage of the indicated DNA constructs by MEF, HeLa, and HT-29 by nuclear extracts obtained from cells grown in the absence and presence of 5-FU. Activity measurements were performed in the presence and absence of UGI to determine the contribution of UNG to the observed cleavage activity. The identical ssU activities of extracts prepared from cultures grown in the absence and presence of 5-FU indicates that UNG is not down regulated in the presence of the drug.