The antimetabolites 5-fluorouracil (5-FU)1
, 5-flurodeoxyuridine (5-dUrd) and raltitrexed (RTX) are widely used for the treatment of colorectal, breast, and head and neck cancers (1
). Fluoropyrimidines are metabolized much like uracil and deoxyuridine and can be enzymatically converted to the active metabolite 5-FdUMP (). A binary complex between 5-FdUMP and 5,10-methylenetetrahydrofolate irreversibly inhibits thymidylate synthase (TS), blocking de novo
production of dTMP and also resulting in accumulation of dUMP. The resulting thymine nucleotide pool deficiency brought about by fluoropyrimidine drugs was originally thought to induce the therapeutic effect by a process called “thymineless death”. However, an additional hallmark of fluoropyrimidine treatment is the polymerase catalyzed incorporation of dUMP and 5-F-dUMP into DNA resulting in U/A, 5-FU/A and 5-FU/G base pairs which are substrates for various uracil DNA repair glycosylases (1
). Similarly, RTX is a TS-specific folate mimic that also prevents TMP synthesis, but unlike fluoropyrimidines, RTX only results in the accumulation of dUMP and U/A base pairs in DNA. Thus, the toxicity mechanisms of these drugs will depend on the dUTP, 5-F-dUTP and TTP pool levels, as well as the relative specificities of the cellular uracil DNA glycosylase activities towards these uracil containing base pairs.
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 (more ...)
Several previous studies have investigated which DNA glycosylase enzymes are responsible for excising uracil and 5-FU bases in the context of U/A, 5-FU/A and 5-FU/G base pairs, and which are responsible for the toxic effects of the drug (6
). In a study performed in budding yeast, Seiple and coworkers showed that deletion of the base excision repair enzyme uracil DNA glycosylase (UNG) resulted in a huge accumulation of U in the yeast genome during treatment with 5-FU (~ 4% of genomic thymidine levels), and that ung
- yeast were protected
against the cytotoxic effects of 5-FU (8
). This study thus established two mechanistic aspects of 5-FU toxicity in the yeast system: (i) 5-FU treatment results in elevated dUTP and accumulation of U in genomic DNA, and (ii) 5-FU toxicity is dependent on excision of U by yeast UNG (UNG is the only enzyme in yeast that removes uracil from DNA).
In contrast to the yeast findings, 5-FU toxicity studies using ung+
mouse embryonic fibroblasts (MEFs) indicated that UNG was not involved in the toxicity mechanism (7
). Since there are four different DNA glycosylases in both mice and humans capable of excising U and 5-FU from DNA (UNG, SMUG1, TDG, MBD42
), it is perhaps not surprising that the role of yeast UNG could be supplanted by the combined activities of SMUG1 and TDG in MEF cells (9
). From these combined studies, distinct roles for UNG, TDG and SMUG were suggested. UNG was indicated to have no role in preventing or precipitating the toxic effects of 5-FU in MEFs (7
), a conclusion also obtained in another study using RTX and HEK293 cells (13
). In the MEF cell studies, the glycosylase activity of TDG precipitated 5-FU toxicity in a manner analogous to UNG in yeast, while 5-FU and U excision by SMUG1 was indicated to be protective against 5-FU toxicity.
To further understand these complex findings, we now examine the extent to which fluoropyrimidines and RTX alter the dUTP and TTP nucleotide pools in human, mouse and DT40 chicken cells, and we measure the specific activities of the DNA glycosylases hUNG2, hSMUG1 and hTDG against the diverse lesions that are generated from these drugs. In human and mouse cells, we find only small increases in the dUTP/TTP ratio after drug treatment in standard media, but much larger increases using dialyzed media that is depleted in folate and thymidine. Thus previous studies using standard media may not have induced high dUTP levels, and consequently, genomic uracil incorporation during S phase would have been limited. In contrast, 5-FdUrd and RTX induce a large increase in the dUTP/TTP ratio DT40 chicken cells. Despite UNG being the only U/A glycosylase activity in DT40 cells, the chicken UNG was found to provide no protective effect against the toxic effects of RTX, similar to results with human cells. Although UNG action is curiously irrelevant to drug toxicity in both human and chicken cells (vide supra), in vitro kinetic analyses of purified hUNG2, hSMUG1 and hTDG, as well as activity measurements using nuclear extracts, established that hUNG2 is paradoxically the primary cellular activity that removes U and 5-FU from DNA. We suggest that human and chicken cells are tolerant to U/A base pairs at the density they are introduced during drug treatment, and that the repair activity of UNG during S phase is masked by this tolerance. The UNG independent toxicity of the TS drugs with DT40 cells indicates that the killing mechanism is independent of both uracil base excision repair and mismatch repair (which acts on 5-FU/G pairs).