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Cancer Research 67, 940, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-2960
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

5-Fluorouracil Incorporated into DNA Is Excised by the Smug1 DNA Glycosylase to Reduce Drug Cytotoxicity

Qian An, Peter Robins, Tomas Lindahl and Deborah E. Barnes

Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom

Requests for reprints: Tomas Lindahl, Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, United Kingdom. Phone: 44-17-0762-5993/5995; Fax: 44-20-7269-3803; E-mail: tomas.lindahl{at}cancer.org.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
5-Fluorouracil (FU) has been widely used for more than four decades in the treatment of a range of common cancers. The fluorine-substituted uracila analogue is converted to several active metabolites but the mechanism of cytotoxicity has remained unclear. In a widely cited but unsubstantiated model, FU is thought to kill cells via the inhibition of thymidylate synthase and increased use of dUTP in place of TTP during DNA replication, with subsequent excision of high levels of uracil causing the fragmentation of newly synthesized DNA. Using gene-targeted cell lines defective in one or both of the two mammalian uracil-DNA glycosylase repair enzymes, we were able to test this model of FU cytotoxicity. Here, we show that incorporation of FU itself into DNA has been previously underestimated and is a predominant cause of cytotoxicity. FU readily becomes incorporated into the DNA of drug-treated cells, and accumulation of FU in the genome, rather than uracil excision, is correlated with FU cytotoxicity in mammalian cells. Furthermore, the Smug1, but not the Ung, uracil-DNA glycosylase excises FU from DNA and protects against cell killing. The data provides a clearer understanding of the action of FU, suggesting predictive biomarkers of drug response and a mechanism for acquired resistance in tumors. [Cancer Res 2007;67(3):940–5]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
5-Fluorouracil (FU) is widely used in the treatment of a range of cancers, particularly colorectal cancer but also cancers of the breast, head-and-neck, and aerodigestive tract (1). After four decades in clinical use, FU or an oral prodrug (2) is presently used in the treatment of some two million individuals a year (3). The fluorine-substituted uracil analogue is an antimetabolite that was designed to inhibit DNA synthesis in an early example of the rational development of a cytotoxic drug (4). Inhibition of thymidylate synthase (TS) by FdUMP prevents the conversion of dUMP to the sole de novo source of dTMP (Fig. 1 ), depleting the cell of dTTP for use in DNA replication and repair. This leads to deoxynucleoside triphosphate precursor pool imbalances and increased levels of dUTP. In a frequently proposed model, FU-mediated cell death is attributed to the use of dUTP in place of dTTP during DNA synthesis, and subsequent DNA fragmentation due to extensive uracil excision in newly synthesized DNA or repeated futile repair attempts in the presence of a high dUTP/dTTP ratio (reviewed in ref. 1). TS status has been correlated with tumor response (5), and the effects of modulating cellular levels of dUTPase (Fig. 1) would seem to be consistent with the model (6, 7). However, mammalian cells lacking the major replication-associated uracil-DNA glycosylase, Ung (8), do not show altered resistance to FU (9), and Ung overexpression does not affect the sensitivity to specific TS inhibitors (10). Thus, the model that FU killing is mediated through inhibition of TS and increased incorporation of uracil in DNA remains unsubstantiated.


Figure 1
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  		Figure 1. FU metabolism and the role of Smug1. FU is converted to the active precursors FUTP, FdUTP, and FdUMP (boldface); these can be incorporated into RNA, DNA, or inhibit TS, respectively. The key steps associated with FU sensitivity (red) or FU resistance (green). DHFU, dihydrofluorouracil. Dashed lines indicate our main findings here: (a) incorporation of FU into DNA is cytotoxic; (b) Smug1 excises FU from DNA in vivo and protects cells from FU killing.

 
FU is converted to several active metabolites (Fig. 1). In addition to inhibiting TS, FU may exert its effects through incorporation of the fluoronucleotide into RNA and disruption of RNA processing (reviewed in ref. 1). FU is also incorporated into DNA following its conversion to FdUTP, either via FdUMP or by reduction of the ribonucleoside diphosphate. The introduction of FU into DNA via the latter pathway may be unaffected by FdUMP-mediated inhibition of TS (Fig. 1). dUTPase hydrolyses FdUTP as well as dUTP (11), and the significance of FU incorporation in DNA has been difficult to evaluate from the low levels measurable in drug-treated normal or tumor cells (12–15). Interestingly, both the Ung and Smug1 uracil-DNA glycosylases have been reported to excise FU from DNA in vitro (16). Smug1 is not found in lower eukaryotes such as Saccharomyces cerevisiae and has only evolved as a second uracil-DNA glycosylase in vertebrates (17), in which it is nonredundant with the Ung enzyme in excising uracil or its derivatives from the mammalian genome (8, 18, 19). Thus, FU toxicity in S. cerevisiae would not be a relevant model for cancer chemotherapy (20). Studies in mammalian cells show that FU cytotoxicity is not mediated by Ung-initiated excision of uracil from DNA (9) but have not addressed the role of Smug1. Here, using gene-targeted cell lines defective in one or both of the mammalian uracil-DNA glycosylases, for the first time, we were able to show the contribution of FU incorporation in DNA to FU cytotoxicity and the relevance of the Smug1 uracil-DNA glycosylase in protecting against cell killing (Fig. 1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The Ung–/– mouse embryo fibroblast (MEF) cell line, isogenic wild-type (Ung+/+) control, and stable small interfering RNA–mediated knockdown of Smug1 in both a Ung+/+ (Smug1{downarrow} cell line) and a Ung–/– background (Smug1{downarrow}, Ung–/– cell line) have been described (19). Cells were cultured in DMEM/Ham's F12 (3:1) with 10% fetal bovine serum.

High-performance liquid chromatography analysis of bases released from the DNA of FU-treated cells by recombinant Smug1 and Ung. Exponentially growing cells (~107) were treated with FU-2-14C (53 mCi mmol–1; Sigma, St. Louis, MO), treated with nonradiolabeled FU and labeled with uracil-2-14C (52 mCi mmol–1; Sigma), or treated with nonradiolabeled FU. The concentration of FU (and uracil) used was 10 µmol/L and cells were exposed to FU for 48 h (≤two cell divisions). Genomic DNA was isolated from the cells (Wizard Genomic DNA purification kit; Promega, Madison, WI) and 10 µg 14C-labeled DNA or 15 µg nonradiolabeled DNA was incubated with recombinant human SMUG1 and UNG in standard enzyme reactions (see below). After incubation at 37°C for 1 h, DNA was precipitated with ethanol and the supernatant analyzed by high-performance liquid chromatography (HPLC), as described previously (19). Fractions were collected at 0.5 min intervals, and released DNA bases detected by scintillation counting or UV absorbance at 254 nm; reference compounds were detected by UV absorbance.

In vitro DNA glycosylase assays. Standard uracil-DNA glycosylase assays were carried out using recombinant human SMUG1 or UNG proteins in 20 mmol/L of Tris-HCl (pH 8.0), 50 mmol/L of NaCl, 1 mmol/L of DTT, 1 mmol/L of EDTA, 100 µg mL–1 bovine serum albumin, 1 ng of AP endonuclease, and 0.24 pmol of DNA substrate at 37°C for 1 h, as described previously (19). The DNA substrate contained a centrally placed U:A or FU:A base pair in the 5'-32P-end–labeled strand of a 19-mer double-stranded oligonucleotide. After the addition of 1 mol/L of piperidine and incubation at 90°C for 20 min, samples were dried under vacuum, denatured in 95% formamide, and analyzed by 10% PAGE and phosphorimaging. Oligonucleotides were synthesized by MWG-Biotech;1 the FU-containing oligonucleotide was HPLC-purified and certified by MALDI-TOF mass spectrometry.

FU cell survival assays. Exponentially growing cells plated in triplicate in 100 mm dishes were treated at a range of drug doses with FU or fluorodeoxyuridine (FUdR), and surviving colonies were scored after 10 to 12 days in culture (as a percentage of the untreated control and after adjustment for plating efficiency), essentially as described (19). Where cells were supplemented with thymidine, 100 µmol/L of thymidine was given along with 1 µmol/L of deoxycytidine (21). FU, FUdR, and thymidine were obtained from Sigma.

Generation of stable Smug1-overexpressing MEF cell lines. The murine Smug1 open reading frame (837 bp; ref. 17) was cloned under the control of the cytomegalovirus promoter in the pTARGET Mammalian Expression System (Promega). The construct was linearized with ScaI and transfected into the wild-type MEF cell line using Polyfect Transfection Reagent (Qiagen, Chatsworth, CA). G418-resistant colonies were isolated following culture in 1.4 mg mL–1 of G418 and the level of Smug1 expression was assayed by quantitative real-time PCR of reverse-transcribed total RNA, as described previously (19). Colonies showing elevated Smug1 mRNA levels were subcultured for three to four passages in G418, and Smug1 expression was re-assessed and confirmed by Western blotting with antibodies against human SMUG1 (17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Accumulation of FU in the DNA of FU-treated Smug1-deficient cells. We have previously generated a Ung–/– MEF cell line (8) and stable small interfering RNA–mediated knockdown of Smug1 (Smug1{downarrow}) in both the Ung–/– and Ung+/+ background (19). In order to determine whether FU is incorporated and persists in the DNA of drug-treated uracil-DNA glycosylase-deficient cells, the Ung–/– cell line, Smug1{downarrow} and Smug1{downarrow}, Ung–/– cell lines were exposed to 14C-labeled FU, genomic DNA was isolated from live cells after 48 h of FU exposure, and samples were digested with recombinant Smug1 and Ung. Radiolabeled ethanol-soluble products released by the recombinant proteins were then resolved by HPLC and identified by comparison with reference compounds (Fig. 2A ). HPLC analysis resolved a small discrete peak of radioactive material in DNA from wild-type cells that was coincident with a FU marker; a similar small peak of 14C-FU was detected in Ung–/– cells. This corresponded to 1.5 x 105 FU residues per diploid genome in both wild-type and Ung–/– cells (Table 1 ). In marked contrast, a large peak of 14C-FU was detected in DNA from both the Smug1{downarrow} and Smug1{downarrow}, Ung–/– cell lines, corresponding to 4.3 x 106 and 3.2 x 106 FU residues per diploid genome, respectively (Table 1); a slightly lower level of FU incorporated in the double mutant may reflect the slower doubling time of this cell line (data not shown) and the limited labeling period. The data shows that there is substantial incorporation of FU in the DNA of drug-treated cells, with no effect of Ung deficiency on FU levels but a 20- to 30-fold increase in the level of FU in the genome of Smug1-deficient cells (Fig. 2A; Table 1); this is equivalent to the substitution of ~1 in 500 thymine residues.


Figure 2
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  		Figure 2. Accumulation of FU and/or uracil in the genome of FU-treated cells. Cells were treated for 48 h with 10 µmol/L of FU (and uracil) as follows: 14C-FU (A), nonradiolabeled FU and 14C-uracil (B), and nonradiolabeled FU (C). Genomic DNA was isolated and incubated with recombinant human SMUG1 and UNG proteins. Ethanol-soluble material was analyzed by HPLC, and released DNA bases detected by scintillation counting (A and B) or UV absorbance at 254 nm (C) in comparison with reference compounds. A and B, wild-type ({blacksquare}), Ung–/– ({blacktriangleup}), Smug1{downarrow} ({square}), and Smug1{downarrow}, Ung–/– ({triangleup}) cell lines; C, Smug1{downarrow}, Ung–/– cell line.

 

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  		Table 1. Incorporation of FU and U in the genome of uracil-DNA glycosylase-deficient cell lines

 
Similar experiments were conducted in which cells were treated with the same molar concentration of FU as above but this was nonradiolabeled; cells were instead labeled with 14C-uracil in order to examine the levels of uracil incorporated into the DNA. These experiments showed that uracil accumulates in cellular DNA of FU-treated cells, but to a lesser degree than FU (Fig. 2B), and in wild-type cells, there were 8.2 x 104 uracil residues per diploid genome (Table 1). However, unlike FU in DNA, uracil levels were affected by the loss of either Ung or Smug1. Thus, there was an increase of uracil in DNA in Ung–/– as well as Smug1{downarrow} cells, and an additive 10-fold increase (8.2 x 105 uracil residues per diploid genome) in the double mutant. The accumulated level of uracil was 3- to 4-fold less than that of FU in the Smug1{downarrow}, Ung–/– cell line (Table 1), representing the substitution of ~1 in 2,000 thymine residues. Both FU and U were also detected, by UV absorbance in comparison with known quantities of reference standards, in Smug1{downarrow}, Ung–/– cells treated with nonradiolabeled FU, and were present at levels equivalent to those observed in radiolabeling experiments (Fig. 2C). When total RNA as well as genomic DNA was isolated from duplicate samples of FU-treated cells, the ratio of 14C-FU (cpm µg–1) incorporated into RNA/DNA in the wild-type and Ung–/– cell lines was ~11:1, in agreement with other studies (15), and this ratio was reduced in the Smug1{downarrow} and Smug1{downarrow}, Ung–/– cell lines, consistent with increased levels of FU in the DNA of Smug1-deficient cells (data not shown). Our data indicate that whereas both Smug1 and Ung excise uracil incorporated in U:A base pairs in vivo, the Smug1 uracil-DNA glycosylase—but not Ung—excises FU arising through the use of FdUTP instead of dTTP during DNA synthesis (Fig. 1).

In vitro DNA repair assays. In vitro, the Ung and Smug1 uracil-DNA glycosylases efficiently excise uracil from U:A base pairs in nucleosomes as well as in naked DNA (22). Both enzymes have also been reported to excise FU from DNA in vitro (16). In light of our data demonstrating the accumulation of FU in the DNA of Smug1- but not Ung-deficient cells, repair assays were conducted to examine the FU-excision activity of Smug1 and Ung in vitro. We assayed the relative activity of each enzyme on a double-stranded oligonucleotide substrate containing a centrally placed FU:A or a U:A base pair. Both enzymes are considerably less active on a FU:A–containing versus a U:A–containing substrate but, whereas Ung activity is ≥100x reduced, Smug1 activity is only ~10x reduced on a FU:A versus U:A base pair (Fig. 3 ). FU:A excision activity could not be detected in wild-type cell-free extracts (data not shown) so it was not possible to assess the contribution of Smug1 and Ung to the total cellular FU-excision activity. However, the greatly reduced activity of recombinant Ung on FU:A versus U:A in vitro (Fig. 3) correlates with the failure of Ung to contribute to FU repair in vivo (Fig. 2A; Table 1). Conversely, the much more robust activity of recombinant Smug1 on FU:A base pairs is consistent with the accumulation of FU in the DNA of Smug1-deficient FU-treated cells and the conclusion that Smug1 excises FU incorporated into FU:A DNA base pairs in vivo.


Figure 3
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  		Figure 3. In vitro activity of the Smug1 and Ung enzymes on FU:A DNA base pairs. DNA glycosylase activity of recombinant human SMUG1 or UNG proteins was assayed on a 19-mer double-stranded oligonucleotide substrate containing a centrally placed U:A or FU:A base pair in the 5'-32P-end–labeled strand, as indicated. After cleavage of the resultant abasic site, the 9-mer 32P-labeled product was resolved by PAGE.

 
Sensitivity of Smug1-deficient cell lines to FU killing. In order to determine whether the observed accumulation of FU in the genome of Smug1-deficient cells had any effect on cell viability, we compared the sensitivity of the Smug1{downarrow} and Smug1{downarrow}, Ung–/– MEF cell lines to FU killing, with that of the Ung–/– cell line and wild-type control. Smug1-deficiency significantly altered the cellular response to FU (Fig. 4A ) and the Smug1{downarrow} cell line was ~2-fold more sensitive to FU (D37 = 3.2 µmol/L) than the wild-type control (D37 = 6.8 µmol/L; D37 is the drug dose that results in 37% cell survival). In agreement with previous data (9), there was no significant difference in survival of the Ung–/– cell line treated with FU in comparison with the wild-type control. Furthermore, there was no additive effect due to loss of Ung, with Smug1{downarrow}, Ung–/– cells showing the same sensitivity as the Smug1{downarrow} cell line. The relative sensitivity of the four cell lines to killing by FU correlates with the levels of FU measured in cellular DNA; it does not correlate with the levels of uracil in DNA (Fig. 2A; Table 1). FU in DNA was measured after 48 h treatment of cell lines with 10 µmol/L of 14C-FU (Fig. 2A); this same drug dose resulted in 13.5% versus 4.2% survival of wild-type and Smug1{downarrow} cells, respectively (Fig. 4A). Thus, the persistence of FU in DNA, rather than the excision of uracil, is the predominant means of FU-mediated cell killing. Our data show that accumulation of FU in DNA is cytotoxic and base excision repair of FU initiated by Smug1 protects cells from FU killing.


Figure 4
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  		Figure 4. Smug1-deficient cells were hypersensitive to killing by FU. Wild-type ({blacksquare}), Ung–/– ({blacktriangleup}), Smug1{downarrow} ({square}), and Smug1{downarrow}, Ung–/– ({triangleup}) MEF cell lines were treated with FU (A) or FUdR (B) at the doses indicated and surviving colonies scored after 10 to 12 days in culture (as a percentage of the untreated control). Points, mean from three experiments; bars, SE. B, FUdR-treated cells were grown either with (- - -) or without (—) supplementation with 100 µmol/L of thymidine and 1 µmol/L of deoxycytidine. Note the different dose ranges on the X-axes in (A) and (B).

 
As with FU, Smug1{downarrow} and Smug1{downarrow}, Ung–/– cells treated with FUdR showed significantly increased sensitivity to the fluoropyrimidine, whereas there was again no marked difference in the survival of Ung–/– versus wild-type cells (Fig. 4B). Thus, Smug1-deficient cells (D37 = 11.6 µmol/L) were ~2-fold more sensitive to FUdR than wild-type cells (D37 = 20.8 µmol/L). This indicates that FU is introduced into the DNA by treatment with FUdR as well as FU, and hypersensitivity of Smug1{downarrow} cells to FUdR is, as with FU treatment, due to the deficiency in Smug1-initiated base excision repair of FU incorporated in place of thymine in DNA. The cell is able to synthesize dTMP de novo via a nonessential salvage pathway catalyzed by thymidine kinase, bypassing the effects of TS inhibition and restoring the dTTP pool; due to the allosteric regulation of deoxynucleoside triphosphate synthesis, cells were supplemented with thymidine as well as a trace of deoxycytidine (21). Thymidine supplementation may not affect the use of FdUTP in DNA synthesis (Fig. 1). Consistent with this, supplementing FUdR-treated cells with thymidine decreased the sensitivity of all four cell lines but Smug1{downarrow} cells still remained more sensitive than Ung–/– and wild-type cells (Fig. 4B).

Both wild-type and Smug1-deficient cell lines were 3- to 4-fold more sensitive to killing by FU than FUdR (note different dose ranges on the X-axes in Fig. 4A and B), and although thymidine supplementation also proportionately decreased the sensitivity of both cell lines to FU, it was less effective in rescuing FU-treated compared with FUdR-treated cells (data not shown). These data indicate that FU-mediated killing of wild-type cells is predominantly due to active metabolites that are preferentially produced from FU rather than FUdR; these are likely to be the RNA and DNA precursors, FUTP and FdUTP, rather than the TS-inhibitory FdUMP (Fig. 1).

Overexpression of Smug1 protects cells against FU-mediated cell killing. We have shown that FU incorporation in DNA is a predominant means of FU-mediated cell killing and that cells deficient in DNA base excision repair of FU by Smug1 are sensitized to cell killing. We therefore investigated whether overexpression of Smug1 in wild-type cells might increase cellular resistance to FU killing. A construct expressing murine Smug1 under the control of the cytomegalovirus promoter was transfected into the wild-type MEF cell line. Sublines were established that stably expressed moderately elevated levels of Smug1, as ascertained by quantitative real-time PCR and verified by Western blotting with Smug1-specific antibodies. Two sublines expressing ~1.5x and ~2.5x the wild-type level of Smug1 protein were assayed for cellular sensitivity to FU in comparison with the parental wild-type line (Fig. 5 ). There was a significantly increased resistance to FU in the cell line expressing ~2.5x Smug1 protein relative to the wild-type parental line (D37 ratio 1.7:1).


Figure 5
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  		Figure 5. Overexpression of SMUG1 increases the FU-resistance of wild-type cells. A wild-type MEF cell line ({blacksquare}), and stable transfected clones overexpressing recombinant SMUG1 protein, at ~2.5-fold (bullet) or ~1.5-fold ({circ}) wild-type levels, were treated with FU at the doses indicated and surviving colonies scored (as a percentage of the untreated control), as in Fig. 4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that the Smug1 DNA glycosylase excises FU from DNA in vivo; extensive accumulation of FU in the genome of Smug1-deficient cells sensitizes them to FU killing, providing direct proof that FU incorporated in DNA is cytotoxic (Fig. 1). The level of FU in DNA measured here in wild-type cells is comparable to early reports for normal or tumor cell lines in which the occurrence of FU in DNA was first described (12–15). However, the extent to which FU becomes incorporated in DNA and its relevance to FU cytotoxicity is only revealed here in a Smug1-deficient cell line (Fig. 2A). There is an appreciable level of FU in DNA even in the presence of Smug1 (Table 1), and it seems likely that this would also contribute to the toxicity of FU in wild-type cells. Consistent with this, a modest ~2.5-fold increase in the level of Smug1 protein significantly increased the resistance of FU-treated cells to killing. Thus, the excision of FU from DNA by Smug1 may be relatively inefficient, in agreement with the limited FU excision capacity observed in normal mammalian cells (23). We have shown in in vitro assays that Ung has greatly reduced activity on FU:A versus U:A base pairs, which would explain why Ung does not contribute to FU repair in vivo. Furthermore, as Ung but not Smug1 is sorted to the mitochondria as well as the nucleus (24), this would leave the mitochondrial genome vulnerable to incorporated FU and a possible target of FU toxicity in wild-type cells. SMUG1 is able to excise uracil derivatives with substitutions at the C5 position of the pyrimidine ring, as the aromatic residue that forms a steric barrier to binding of C5-modified bases in UNG is not conserved in the active site structure of SMUG1 (18). Our data are consistent with this, demonstrating considerably less discrimination by Smug1 than Ung against FU:A versus U:A base pairs in vitro, and the contribution of Smug1 but not Ung to FU repair in vivo (Fig. 4).

Smug1-deficient cells are sensitized to killing by FU, with no effect of Ung on FU survival. Thus, FU cytotoxicity is directly correlated with the loss of Smug1 FU-excision activity and greatly increased levels of FU in DNA. The accumulation of uracil in the DNA of Smug1{downarrow} cells as well as Ung–/– cells, with an up to 10-fold increase in the Smug1{downarrow}, Ung–/– double mutant, was not correlated with FU survival. As Smug1{downarrow}, Ung–/– cells lacking uracil-DNA glycosylase activity are sensitized to killing, the data clearly oppose a model in which FU toxicity is due to the excision of uracil from DNA. Our data do not directly address whether specific inhibition of TS kills cells due to elevation of the dUTP precursor pool and associated base excision repair of uracil in DNA, but the FU hypersensitivity of Smug1{downarrow} cells versus unaltered resistance of Ung–/– cells shows that use of FdUTP in DNA synthesis rather than FdUMP-mediated inhibition of TS is the predominant mechanism by which FU kills cells. Consistent with this, all MEF lines here were considerably less sensitive to FUdR than to FU, as has been described for other murine and human cell lines (15). FUdR would be expected to exert its effects primarily through TS inhibition, as FUdR is converted directly to FdUMP, whereas the conversion of FU to FUMP allows incorporation in RNA but also DNA via reduction of the ribonucleoside diphosphate, which would be unaffected by TS inhibition (Fig. 1). Thus, supplementing cells with thymidine to overcome TS-inhibition and restore the dTTP pool (Fig. 1) only partially rescued FU-treated wild-type cells and did not reduce the hypersensitivity of Smug1-deficient cells to either FU or FUdR, as the use of FdUTP in DNA synthesis remained unaltered.

It is presently unclear why FU in DNA is cytotoxic. We cannot formally exclude the possibility that FU bases themselves are benign substitutions in DNA but that instead a small proportion are converted to a highly toxic lesion in situ. However, massive substitution of ~1 in 500 thymine residues with FU in the genome of Smug1-deficient cells (Table 1) would itself be expected to affect fundamental processes, such as the transcription of essential genes or the establishment of the chromatin structure, by altering DNA dynamics in A:T–rich regions (25, 26) or interfering with protein-DNA interactions (27). Such processes might also be compromised if there were nonproductive binding to FU residues in DNA by another DNA repair/processing enzyme. Mismatch repair (28), and the MBD4 and TDG mismatch-specific DNA glycosylases (29, 30), have also been reported to act on FU in vitro but in the context of a FU:G mispair; use of FdUTP during DNA synthesis typically gives rise to an FU:A base pair (25) but rare mutagenic FU:G mispairs could theoretically arise in vivo (26, 31). In contrast to Smug1-deficient cells, and by an as yet unexplained mechanism, Mbd4–/– MEFs (29), and mismatch repair–defective cell lines and tumors (28) are apparently resistant to FU. Thus, rare FU:G base pairs might arise upon replication of FU:A–containing DNA and be abortively processed by mismatch repair or a mismatch-specific DNA glycosylase. Elevated levels of FU in DNA did not seem to be associated with increased DNA fragmentation in FU-treated Smug1-deficient cells (data not shown). TDG, which is a very minor uracil-excision activity compared with UNG or SMUG1 (8), has been reported to act on FU:A in vitro (30); if this is relevant in vivo and whether ablation of Tdg might modify the effect of Smug1-deficiency remains to be determined.

Our data show that the introduction of FU into DNA and its contribution to FU-mediated cell killing has been previously underestimated. This is particularly relevant as formation of FdUTP and its use in DNA synthesis seems to occur irrespective of TS inhibition and the level of the TTP pool in FU-treated cells (Fig. 4), being more clearly correlated with ribonucleotide metabolism and dependent on the activity of ribonucleotide reductase converting FUDP to FdUDP (Fig. 1). The relationship of FU in DNA to both the strategy and efficacy of FU treatment in patients should be further evaluated (2, 3, 32). Our data shows that even a 2- to 3-fold increase in Smug1 protein significantly increases the resistance of FU-treated cells to killing, such that Smug1 up-regulation could provide a previously unrecognized mechanism of acquired drug resistance in tumors. This may be particularly relevant where FU is used in combination with radiotherapy (3), as Smug1 also helps protect cells against killing by low-level ionizing radiation (19). Gene products identified to date as markers of FU resistance have invariably been involved in metabolizing the drug or modulating TS inhibition, rather than directly in repairing FU DNA damage (Fig. 1), but it would now seem pertinent to analyze the Smug1 enzyme during prolonged FU exposure and in gene expression profiles of FU-resistant tumors (33, 34).


   Acknowledgments
 
Grant support: Cancer Research UK.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
1 http://www.mwg-biotech.com/html/all/index.php. Back

Received 8/ 9/06. Accepted 11/20/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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