DNA mismatch repair (MMR)-dependent 5-fluorouracil cytotoxicity and the potential for new therapeutic targets

Abstract

The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.

Figure 1

Metabolism of 5-fluorouracil (FUra) to DNA- and RNA-level metabolites. Developed in the late 1950s and studied intensively over the next 40 years, FUra is still a key chemotherapeutic agent used in the treatment of colon cancer, as well as in adjuvant therapies for a variety of other cancers. Upon entering the cell, FUra is rapidly converted to both 5-fluorouridine (FUrd) and 5-fluoro-2'deoxyuridine (FdUrd) antimetabolites by phosphorylases that add on deoxyribose or ribose units, depending on available substrate ribo- or deoxyribo-nucleosides. Once formed, FUrd or FdUrd are phosphorylated by uridine or thymidine kinases (UK or TK), respectively, to retain the antimetabolites in the cell. Basically, all of the FP-antimetabolites are better substrates than the normal metabolites for each enzymatic step. In general, metabolism of FUra to RNA-level antimetabolites (level 1) leads to less antitumour activity and more general toxicity to normal tissue, as the levels of enzymes that metabolize these RNA-level antimetabolites are not elevated in tumour versus normal tissue. In contrast, enzymes [e.g. thymidine kinase (TK) and thymidylate synthase (TS)] that metabolize DNA-level FUra antimetabolites are elevated in tumour above normal tissue (levels 2, 3). The contribution of FdUrd incorporated into DNA to antitumour activity has been misunderstood and greatly under-estimated. Enzyme abbreviations: UP, uridine phosphorylase; UK, uridine kinase; RNAP, RNA polymerase; rR, ribonucleotide reductase; DNAP, DNA polymerase; TP, thymidine phosphorylase; OPRT, orotic acid phosphoribosyl transferase. Adapted from Meyers et al. (2003).

Figure 2

Minimal mismatch repair uniquely requires MutS homologue(s) (MSH), MutL homologue(s) (MLH/PMS) and an Exonuclease (Exo). E. coli (gram-negative enteric bacteria) MMR (red) uniquely requires a hemi-methylated Dam site (-CH3), the MutH endonuclease, and the MutU (UvrD) helicase. The excision tract (250–1000 bp) extends uniquely from the strand scission to just past the DNA mismatch lesion.

Figure 3

Models for Mismatch Repair. See text for description.

Figure 5

Sensitization of MSI⁺, hMLH1^- RKO6 cells using FdCyd. RKO6 cells are wild-type for the hMLH1 gene, yet are MSI⁺, and lack expression of hMLH1 protein because of hypermethylation of its promoter. RKO6 cells were synchronized by low serum medium and released by re-plating into 10% fetal calf serum-containing medium. Cells were then mock-treated (untreated cells) or exposed to 1 µM FdUrd or FdCyd for 62 h, past the p53 checkpoint as described (Meyers et al., 2001; ;). Cells were then monitored for cell cycle checkpoint responses and changes in the G₂ cell population were graphed over time (h) after release. Note that FdCyd treatment caused G₂ arrest with corresponding hMLH1 expression (not shown). Dashed line, mock-treated synchronized cells; Open squares, FdUrd (1.0 µM, 4 h); Closed circles, FdCyd (1.0 µM, 4 h). Western blotting demonstrated hMLH1 expression and hPMS2 stabilization after FdCyd, but not after FdUrd, exposures. hMLH1, human MutL homologue-1.

Figure 4

Potential use of 5-fluoro-2′-deoxycytidine (FdCyd) for treatment of hMLH1-, MMR-deficient sporadic cancers. FdCyd, which is not a substrate for RNA-level metabolism (e.g. dThyd or Urd phosphorylases), can be manipulated for increased incorporation into DNA by using the dCyd or dCMP deaminase (CD or dCMPD respectively) inhibitors, tetrahydrouridine (H₄Urd) or deoxytetrahydrouridine (dH₄Urd) respectively. Once incorporated, FdCyd can lead to hypomethylation of DNA and re-expression of hMLH1 in MSI⁺ sporadic cancers that are genetically wild-type for hMLH1, but lack hMLH1 expression because of hypermethylation of its promoter, as described by Veigl et al., 1998. Adapted from Meyers et al. (2003). hMLH1, human MutL homologue-1.