Incorporation of dUMP into DNA is a major source of spontaneous DNA damage, while excision of uracil is not required for cytotoxicity of fluoropyrimidines in mouse embryonic fibroblasts

Sonja Andersen2,*, Tina Heine3,*, Ragnhild Sneve2, Imbritt König2, Hans E. Krokan2, Bernd Epe3 and Hilde Nilsen1,4,5

1 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK, 2 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Medical Faculty, N-7489 Trondheim, Norway and 3 Institute of Pharmacy, University of Mainz, D-55099 Mainz, Germany

5 To whom correspondence should be addressed Email: hilde.nilsen{at}biotek.uio.no


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Uracil may arise in DNA as a result of deamination of cytosine or through incorporation of dUMP instead of dTMP during replication. We have studied the steady-state levels of uracil in the DNA of primary cells and mouse embryonic fibroblast (MEF) cell lines from mice deficient in the Ung uracil-DNA glycosylase. The results show that the levels of uracil in the DNA of Ung–/– cells strongly depend on proliferation, indicating that the uracil residues originate predominantly from misincorporation during replication. Treatment with 5-fluoro-2'-deoxyuridine (5-FdUrd) or 5-fluorouracil (5-FU) gives rise to a dose-dependent increase of uracil in Ung–/– MEFs (up to 1.5-fold) but not in wild-type cells. Interestingly, Ung–/– MEFs accumulate AP-sites as well as uracil in response to 5-FdUrd but not to 5-FU. This accumulation of repair intermediates suggests a loss of tightly co-ordinated repair in the absence of Ung, and correlates with stronger inhibition of cell proliferation in response to 5-FdUrd, but not to 5-FU, in Ung–/– MEFs compared with wild-type cells. However, other cytotoxic effects of these fluoropyrimidines are comparable in both wild-type and Ung-deficient cells, demonstrating that excision of uracil from DNA by the Ung uracil-DNA glycosylase is not a prerequisite for obtaining cytotoxicity.

Abbreviations: ConA, Concanavalin A; 5-FdUrd, 5-fluoro-2'-deoxyuridine; 5-FU, 5-fluorouracil; LPS, lipopolysaccharide; MEF, mouse embryonic fibroblast; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PI, propidium iodide; SMUG1, single-strand selective monofunctional uracil glycosylases; TS, thymidylate synthase; UDG, uracil-DNA glycosylase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Spontaneous hydrolytic deamination of cytosine has been estimated to introduce between 100 and 500 uracil residues in the form of U:G mismatches per cell per day (1,2). Recent evidence suggests that uracil is also introduced in a regulated and targeted manner in immunoglobulin loci of mammalian cells by activation induced cytosine deaminase (AID) (3,4). Replicative DNA polymerases incorporate dUMP into DNA with similar efficiency as dTMP (5). The steady-state level of uracil in mammalian DNA is not known but, based on the relative sizes of the substrate dUTP/dTTP pools, it has been calculated that less than one dUMP residue enters DNA per 105 dTMP in normal human lymphoblasts (6). However, the amount of dUMP actually remaining in DNA is even lower due to rapid uracil removal and repair, and a steady-state level of uracil of 10–15 residues per diploid genome has been suggested (7).

Uracil in DNA is recognized and excised by enzymes collectively known as uracil-DNA glycosylases (UDGs). In mammalian cells these include the uracil- (Ung), single-strand selective monofunctional uracil- (Smug1), thymine mismatch (Tdg) and methyl CpG binding domain 1 (Mbd4/Med1)-DNA glycosylases (see ref. 8 for review). Excision of uracil by UDGs initiates the base excision repair (BER) pathway (see ref. 9 for review). Inactivation of BER in mice leads to embryonic lethality (10), suggesting that spontaneous DNA base damage might be formed extensively during development. Although all UDGs are able to remove uracil from DNA, they differ in substrate specificity, expression pattern and intracellular localization. The highly conserved Ung enzyme is the most widely distributed UDG (11). Ung removes uracil from single- as well as double-stranded DNA (12). We have reported previously the generation of gene-targeted mice deficient in the Ung enzyme (13). A modest increase (<1.5-fold) in the global spontaneous mutation frequency in Ung–/– mice (13) was consistent with the presence of a second major UDG activity able to repair deaminated cytosine, contributed by the Smug1 enzyme (14,15). A major distinguishing feature between the Ung and the Smug1 enzymes is the association of Ung with the replication fork via direct interaction with PCNA and RPA (16). Accordingly, the most prominent biochemical phenotype of Ung–/– mice was shown to be an increased steady-state level of uracil in the genome due to a lack of rapid removal of uracil residues incorporated during DNA synthesis (13).

The Ung enzyme was shown recently to be the major UDG responsible for processing U:G lesions induced by AID during somatic hypermutation and class switch recombination in mice (17) and humans (18). Thus, it seems likely that B-cell lymphoma develop in aging Ung–/– mice (19) as a consequence of the central role of Ung in antibody diversification, but the formal possibility that misincorporated uracil in DNA might have adverse effects remains.

Incorporation of dUMP has also been suggested to be one of the mechanisms of cytotoxicity for anticancer drugs that inhibit thymidylate synthase (TS), such as 5-fluorouracil (5-FU) (20). Inhibition of TS by 5-FU is accompanied by an increase in the dUTP/dTTP ratio and incorporation of uracil into DNA (2123). Mechanistically, double-strand breaks may form upon attempted repair of two opposing uracil residues (24). Accumulation of single- (SSB) and double-strand breaks has been verified by alkaline elution of DNA from 5-FU-treated cells and tissues (22,23,25). Reduction of cytotoxicity and strand-break formation upon increased expression of dUTP nucleotidohydrolase/deoxyuridine triphosphatase (dUTPase), which hydrolyses dUTP to dUMP, suggests a mechanistic involvement of uracil incorporation in double-strand break formation (26,27). These models rely on the direct involvement of UDGs in the mechanism of cytotoxicity, and the availability of Ung-deficient mice allows us to address these questions directly.

Here, we have used the comet and the alkaline filter elution assays to distinguish between the relative contribution of spontaneous cytosine deamination and incorporation of dUMP as the most abundant source for uracil in the genome in mouse cells. We also show that Ung–/– and Ung+/+ cells are equally sensitive towards the important anticarcinogenic agent 5-FU, indicating that the cytotoxicity of this agent does not depend on the generation of uracil in DNA. Another fluoropyrimidine, 5-fluoro-2'-deoxyuridine (5-FdUrd), seems to utilize partly different mechanisms as Ung–/– mouse embryonic fibroblast (MEF) cells accumulate both uracil and repair intermediates (AP-sites) concomitant with a growth inhibition that is much higher than in wild-type MEFs.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice and cell lines
The generation of gene-targeted Ung–/– mice has been described previously (13). Primary MEF cultures were established by standard procedures from individual embryonic day 13.5 (E13.5) embryos derived from heterozygous matings of Ung+/– F1 progeny of chimaeric matings. Permanent Ung–/– and Ung+/+ cell lines were established from transformed clones arising spontaneously after repeated passage in culture (28). MEFs were cultured in Dulbecco's modified Eagle's medium high glucose with glutamine/HAM's F12 with sodium pyruvate (1/1), 10% fetal calf serum, 1x non-essential amino acid solution and penicillin/streptomycin. To obtain confluent cells, 106 cells/175 cm2 flask were seeded and cultured with a change of medium every second day. Confluency, defined as no detectable cell doubling in 48 h, was reached after 8 days. Confluent cells were given fresh medium every day for 3 days prior to analysis by the alkaline elution assay.

Quantification of UDG-sensitive sites and AP-sites by alkaline elution
A modified alkaline elution assay in combination with recombinant repair DNA-glycosylases was used to quantify uracil residues, AP-sites and SSB. The assay followed the protocol of Kohn et al. (29) with modifications described previously (30,31). Briefly, cells were collected on a polycarbonate filter and lysed. After extensive washing, the DNA remaining on the filter was treated exhaustively with recombinant UNG{Delta}84 enzyme (32) (up to 100 ng/ml; see Figure 1A) for 50 min at 37°C. Resulting AP-sites were subsequently converted into SSB by incubation with T4 endonuclease V (Endo V), again for 50 min at 37°C. Fractions were collected after elution of DNA from the filter with an elution buffer of pH 12.2. The number of uracil residues incised by the UNG enzyme was obtained by subtraction of the number of AP-sites plus SSB determined in a parallel experiment in which UNG treatment was omitted. Likewise, the number of AP-sites was obtained by correcting the sum of AP-sites and SSB for the number of SSB determined in incubations without repair enzymes. The slope of an elution curve obtained with {gamma}-irradiated cells was used for calibration (6 Gy = 1 SSB/106 bp).



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Fig. 1. Quantification of uracil levels in the genome of Ung-deficient and wild-type cells. Increasing levels of recombinant human UNG{Delta}84 enzyme was used in alkaline elution to determine the number of UNG-sensitive sites in genomic DNA from Ung–/– (filled symbols) and Ung+/+ (open square, at 100 ng/ml UNG{Delta}84 only) MEF cells (A). Similarly, the numbers of uracil residues in the genome of primary cells isolated from 6 to 8 months old Ung+/+ (open bars) and Ung–/– (filled bars) mice were determined by alkaline elution (B) and the comet assay (C). The number of mice tested is given above the bars. Errors are given as standard deviation of the mean.

 
Cell proliferation assay
MEFs were seeded at 1 x 105 cells in 25-cm2 flasks. A reference flask was counted 24 h after seeding to get the number of attached cells. Media in the remaining flasks were replaced with fresh, drug-supplemented media and the cells were cultured for another 48 h. Subsequently, the number of dead cells in the media was determined; the adherent cells were washed with PBS, trypsinized with a standard trypsin–EDTA solution, and counted with a coulter counter.

Isolation of primary cells for alkaline elution
Primary hepatocytes, splenocytes, kidney cells and spermatozoa from Ung–/– and control mice were isolated by published protocols which, in the case of hepatocytes, involved a modified two-step collagenase perfusion technique (33,34).

Isolation of spleen cells for flow cytometry and cell culturing
Spleen cells were isolated according to the protocol from eBioscience. Briefly, the spleen was placed in cold medium (RPMI 1640 supplemented with 10% HI-FCS, penicillin/streptomycin, 0.03% L-glutamine and 5 x 10–5 M mercaptoethanol) immediately after dissection. A single cell suspension was prepared and run through a cell strainer (Falcon, 100 µm). Cells were pelleted (400 g, 4°C, 8 min), re-suspended in 5 ml of Red Blood Cell lysis solution (eBioscience) and incubated at room temperature for 4–5 min with occasional shaking. After the addition of 25 ml PBS, the cells were pelleted, re-suspended in medium and counted. Spleen cells were cultivated in the presence of lipopolysaccharide (LPS, 5 µg/ml, Sigma) and Concanavalin A (ConA, 1 µg/ml, Sigma).

Cell toxicity assays
Cytotoxic effects of TS inhibitors were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates (MEFs at 1500 cells/well, splenocytes at 2 x 105 cells/well). TS inhibitors were added at the time of seeding (splenocytes) or 24 h later (MEFs). After 72-h incubation, medium was removed from MEFs and 150 µl MTT agent (0.5 mg/ml final concentration) was added and incubated for another 4 h at 37°C. 100 µl MTT solution was removed, 100 µl isopropanol/0.04 M HCl added and the plate was left shaking for 1 h prior to reading of absorbance at 570 nM in an ELISA plate reader. For splenocytes, the cells were first re-suspended to resolve aggregates. The MTT stock was added directly to the wells without removing any medium (0.5 mg/ml final concentration), and the cells were incubated as for MEFs. After centrifugation (400 g, room temperature, 8 min), 100 µl of the supernatant was removed and the remaining protocol was as described above for MEFs.

Flow cytometry
Primary spleen cells were re-suspended at 1 x 106 cells/ml in medium containing mitogens. TS inhibitors were added at different concentrations prior to seeding in 96-well plates (2 x 105 cells/well in triplicates). After 72 h, the plates were centrifuged (400 g, 4°C, 8 min) and media carefully removed. The cells were washed once gently in PBS, while taking care not to re-suspend the cells. Fresh PBS (200 µl/well) was added after centrifugation and the cells were re-suspended thoroughly to get single-cell suspensions before transfer to flow tubes containing 500 µl PBS. Two to ten minutes before analysis, 3 µl of propidium iodide (PI) (250 µg/ml stock) was added, and each tube was mixed manually before entering the flow cytometer (Coulter EPICS XL-MCL) to obtain a homogenous distribution of cells in the suspension. All samples were analysed at the same flow rate and for the same time period to obtain relative cell numbers in addition to percentage of PI-stained (dead) cells.

Comet assay
Single-cell suspensions from kidney, spleen, liver and testis were made essentially as described above. After filtering through a 100-µm cell strainer, an aliquot was mixed with 1% low-melting point agarose, embedded and transferred to alkaline lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, pH 10, 1% Triton X-100 added immediately before use). Cultured spleen cells were taken directly from the 96-well plates, re-suspended to resolve aggregates, embedded and lysed. Single-cell gel electrophoresis (comet assay) was performed as described previously (13). Briefly, after embeddeding and lysis (1 h to several days at 4°C), the samples were washed 2x 5 min in cold Ung buffer (20 mM Tris–HCl, pH 7.5, 60 mM NaCl, 1 mM EDTA) and incubated with recombinant human UNG protein (32) (UNG{Delta}84, 1500 ng/slide and 1 h at 37°C for cells used in experiments with TS inhibitors, 150 ng/slide and 20 min for all other cells). Control samples were either left in cold lysis buffer (untreated) or incubated at 37°C with UNG buffer. Electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH>13) was used for DNA unwinding (4°C, 40 min) and electrophoresis (1.2 V/cm, 300 mA, 25 min, 4°C). Samples were neutralized in cold 0.4 M Tris–HCl, pH 7.5 (2x 5 min), briefly dipped in dH2O and dried (50°C, 15 min) before re-hydration (75 µl 1% low-melting point agarose/slide) and staining with ethidium bromide (15 µg/ml, 20 µl/slide). Quantification was done manually by the same observer(s) through all related experiments. All samples were evaluated from two to eight times, organs were in addition evaluated by two observers.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Uracil accumulates in the genome of Ung–/– cells and tissues
Using the semi-quantitative alkaline comet assay, we reported previously that uracil accumulated in the genome of Ung-deficient mice due to slow removal of dUMP residues incorporated during replication (13). In order to get a more accurate measurement of the levels of uracil, AP-sites, and SSB in the genome, we used the alkaline elution assay in combination with repair DNA glycosylases. Purified human UNG enzyme coupled with the AP-lyase T4 Endo V was used to obtain the combined number of uracil residues and AP-sites, whereas AP-sites alone were quantified as EndoV sensitive sites.

Excision of uracil in DNA of Ung–/– MEFs reached saturation at 60–100 ng/ml UNG{Delta}84 enzyme (Figure 1A). Under these conditions the steady-state level of uracil was determined as 0.59 ± 0.10 UNG-sensitive lesions/106 bp, equivalent to ~3600 uracil residues/diploid genome. The levels of AP-sites and SSB were close to the limit of detection (<0.05 sites/106 bp, equivalent to 300 residues/diploid genome). In wild-type MEFs, uracil levels, as well as AP-sites and SSB, were below the detection limit (determined as 0.02 ± 0.05/106 bp). Analysis of these cells using the semi-quantitative comet assay (data not shown and ref. 13) confirmed the alkaline elution results showing an increased steady-state level of uracil in the genome of Ung–/– MEFs. In agreement with the normal development of Ung–/– mice, no adverse effects were observed in growth characteristics of Ung–/– MEFs resulting from the elevated genomic uracil content (data not shown).

Uracil accumulation in cells isolated directly from Ung–/– organs has not been examined previously. Here, significantly increased uracil levels (~900 residues/genome) were found in hepatocytes and splenocytes by alkaline elution of DNA from Ung–/– compared with wild-type mice. In contrast, no significant accumulation was observed in a mixed cell population of mature spermatocytes (Figure 1B). Essentially the same picture was obtained from the comet assay, where significant uracil accumulation was found in spleen (~8-fold) as well as in testis (~9-fold) of Ung–/– compared with wild-type mice (Figure 1C). Here, the testis samples will predominantly reflect the uracil levels in replicating, immature spermatocytes (35). The relative uracil content in liver and kidney was moderately increased (~2-fold), but the absolute level of uracil varied greatly between individual animals (Figure 1C). In summary, the accumulated uracil level was low in tissues with low turnover rates (kidney and liver) while being higher in more actively proliferating cells such as immature sperm cells and, especially, in spleen cells from Ung–/– mice.

Uracil levels in Ung-deficient cells depend on proliferation status
The above experiments suggested that the rate of cell doubling was a major determinant for the genomic uracil content, indicating that misincorporation of dUMP rather than cytosine deamination is the major source of uracil in the genome. If so, proliferating cells would be expected to have higher levels than resting cells. Therefore, a direct test was performed to compare the uracil content in actively cycling and non-cycling MEFs. Compared with exponentially growing Ung–/– MEFs, the uracil content decreases gradually if the cells are kept under conditions of no growth and it stabilizes at ~1400 uracil residues/cell after 8 days of confluence (Figure 2A). However, a uracil content corresponding to the low level seen in wild-type cells is not achieved in confluent Ung–/– cells. This indicates that, given time, complementary repair mechanisms are able to reduce the uracil level. Yet, importantly, they appear unable to fully complement the Ung enzyme in this function. Furthermore, these experiments show that the genomic uracil level is dynamic, increasing rapidly in Ung–/– cells after sub-culture of confluent cells, reaching the steady-state level of exponentially growing cells after 24 h (Figure 2A).



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Fig. 2. Influence of cell proliferation on uracil levels. The uracil content of Ung+/+ (open bars) and Ung–/– (filled bars) MEFs during exponential growth, after 8 days of confluence, and 24 and 72 h after replating of confluent cultures was measured by alkaline elution (A). The proliferative response of Ung+/+ (open bars, n = 3) and Ung–/– (filled bars, n = 3) primary splenocytes from 3-month-old mice was measured in cells grown without stimuli or with LPS (5 µg/ml) and ConA (1 µg/ml). The cells were cultivated for 72 h and pulse-labelled with [3H]thymidine (1 µCi/well) for the last 18 h (B). The level of uracil in the genome of primary splenocytes from 3-month-old Ung+/+ (open bars, n = 3) and Ung–/– (filled bars, n = 3) was measured by the comet assay. Cells were harvested at the time of isolation (no stimuli) and after 72 h of growth in the presence of LPS (5 µg/ml) and ConA (1 µg/ml) (C). Error bars given are standard deviation of the mean.

 
The strong effect of DNA replication on the uracil level can also be measured in primary splenocytes, which in the absence of mitogenic stimulation, proliferate poorly in culture (Figure 2B). After stimulation of proliferation of B- and T-cells with LPS and ConA, the genomic uracil content increased in splenocytes isolated from wild-type as well as Ung–/– mice, with the highest increase seen in Ung–/– cells (Figure 2C). Considering a constant level of the constitutively expressed back-up repair mechanisms in Ung–/– MEFs and splenocytes (13,15), this would imply that the major source of uracil in DNA of mammalian cells is misincorporation of dUMP during replication. The exception appears to be cells in essentially non-cycling tissues, such as mature spermatozoa, where uracil from other sources may dominate. Also, a small contribution from activation of AID in B cells of stimulated splenocytes, causing deamination of cytosine to uracil preferentially in immunoglobulin loci, cannot be excluded.

Accumulation of AP-sites as well as uracil in response to 5-FdUrd in Ung–/– MEFs
The accumulation of uracil in DNA of Ung-deficient cells appears not to affect normal physiology at the cellular level. We were interested to see, however, whether we could increase the genomic uracil content in Ung–/– cells above the steady-state level and whether this would in turn have deleterious consequences for cell physiology. In order to increase the dUTP pool we chose to inhibit TS by treating cells in culture with 5-FdUrd or with the extensively used anticancer agent 5-FU. This is of particular interest, as the manner in which these agents exert their cytotoxic effect is not fully understood.

When wild-type MEFs were exposed to the TS inhibitor 5-FdUrd, we observed no significant increase in the numbers of uracil residues, AP-sites or SSBs compared with untreated controls (Figure 3A, open symbols). In the genome of Ung–/– MEFs, however, the uracil level increased in response to 5-FdUrd from 3600 to a maximum of 5400 uracil residues/diploid genome. Interestingly, whereas AP-sites were undetectable in the absence of TS inhibitors, the level of AP-sites increased dramatically in Ung–/– MEFs in response to 5-FdUrd, reaching 2800 AP-sites/cell at 5 nM 5-FdUrd (Figure 3A, filled symbols). Thus, at these low concentrations of 5-FdUrd (5–10 nM), the number of uracil residues was only twice the number of AP-sites. Higher concentrations of 5-FdUrd did not further increase the number of AP-sites or uracil residues (data >50 nM not shown). As ongoing replication is required for dUMP incorporation, this may be explained by the growth-inhibitory effect of 5-FdUrd. In Ung–/– MEFs, rather low concentrations of 5-FdUrd caused an apparent block of proliferation after exactly one round of replication (i.e. at proliferation factor 2). Wild-type cells proved less sensitive to the growth arrest by 5-FdUrd (Figure 3B).



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Fig. 3. Inhibition of proliferation and accumulation of DNA damage in MEFs after TS-inhibition. Uracil levels (squares), AP-sites (circles) and SSBs (triangles) were measured in response to 5-FdUrd (A) and 5-FU (C) in Ung+/+ (open symbols) and Ung–/– (filled symbols) by alkaline elution. Cell proliferation was measured by counting adherent cells prior to, and 48 h after, treatment with 5-FdUrd (B) or 5-FU (D). The proliferation factor represents the ratio between the number of adherent cells at the end of the experiment and when adding the agent, and is given as a mean of three independent experiments.

 
Similar to 5-FdUrd, 5-FU caused an increase in genomic uracil in Ung–/– but not in wild-type MEFs (Figure 3C). The maximum level of uracil introduced was achieved at 750 nM 5-FU, where there were 6000 uracil residues/cell, which corresponds to the maximum level obtained in response to low concentrations of 5-FdUrd (5–10 nM). Strikingly, there was no accumulation of AP-sites or SSB in response to 5-FU. Thus, although the maximum increase of uracil levels was similar for these drugs they showed differential effects on the total level of DNA damage introduced. In marked contrast to the situation observed with 5-FdUrd, only a slight difference in growth inhibition was seen between Ung–/– and wild-type MEFs in response to 5-FU (Figure 3D). Thus, the qualitative difference between 5-FU and 5-FdUrd was also seen with respect to the cytostatic effects.

Modulation of uracil levels by inhibiting TS in primary splenocytes
The lack of accumulation of uracil in response to TS inhibitors in Ung+/+ MEFs was surprising. Like any transformed cell line, MEFs might carry secondary genetic changes that could alter the response to fluoropyrimidines. As primary cells presumably do not harbour this uncertainty we chose to repeat these experiments using the semi-quantitative comet assay and primary cells isolated from the spleen of Ung–/– and wild-type mice. We chose to analyse primary splenocytes because they showed a highly elevated level of genomic uracil in Ung–/– mice and can be stimulated to grow in cell culture, allowing for incorporation of dUMP into DNA above steady-state levels.

In contrast to wild-type MEFs, genomic uracil levels increased significantly in wild-type splenocytes in response to both 5-FU (Figure 4A, open bars) and 5-FdUrd (Figure 4B, open bars). A maximum level of genomic uracil was observed at 1 µM 5-FU and at 5 nM 5-FdUrd. Note that maximum uracil levels in wild-type cells remained lower than the steady-state levels in stimulated Ung–/– splenocytes (Figure 4A and B, filled bars). The differential response in Ung+/+ MEFs and primary splenocytes indicate that there might be cell type-specific responses to TS inhibition, as suggested previously (36). Contrary to expectation, no significant accumulation was observed in Ung–/– splenocytes in response to either 5-FU (Figure 4A, filled bars) or 5-FdUrd (Figure 4B, filled bars).



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Fig. 4. Accumulation of DNA damage in response to TS-inhibition in primary splenocytes. Uracil accumulation (given as arbitrary units) was measured by the comet assay in mitogen-stimulated primary Ung+/+ (open bars) and Ung–/– (filled bars) splenocytes in response to 5-FU (A) and 5-FdUrd (B). Cell proliferation was measured by flow cytometric counting of the cell number (after PI staining to identify dead cells), after 72 h of culture in the presence of 5-FU (C) or 5-FdUrd (D), and is given as the number of living cells relative to untreated control.

 
This was not a secondary effect of differences in response to mitogenic stimulation, illustrated by similar incorporation of thymidine after pulse labelling (Figure 2B). As uracil incorporation depends on ongoing replication, we measured the proliferation rate of wild-type and Ung–/– primary splenocytes after mitogenic stimulation in the presence of TS inhibitors. After 72 h of culture, the number of living cells was measured by flow cytometry and given relative to untreated controls for 5-FU (Figure 4C) and 5-FdUrd (Figure 4D). Higher levels of inhibition were observed at lower concentrations of 5-FdUrd compared with 5-FU, but the two genotypes experienced a similar degree of proliferation inhibition in response to either drug. Thus, the apparent lack of uracil accumulation in Ung–/– splenocytes could not be ascribed a preferential block of proliferation compared with wild-type splenocytes.

Induction of cell death by TS inhibitors does not depend on Ung
Cell death and DNA fragmentation in response to TS inhibition has been hypothesized to be initiated by UDG-mediated uracil excision. As Ung is the major replication associated UDG in mammalian cells, and therefore the major candidate to mediate this response, we tested whether loss of Ung activity affected the induction of cell death by 5-FU and 5-FdUrd. Similar toxicity was observed for Ung–/– and Ung+/+ stimulated primary splenocytes in response to 5-FU (Figure 5A) and 5-FdUrd (Figure 5B) when analysed for the loss of membrane integrity by PI staining and subsequent flow cytometry. Similar results were obtained using the MTT assay, in which the metabolic activity of the cells is measured (data not shown). Moreover, the two genotypes showed similar response to either drug, suggesting that the Ung enzyme is neither promoting nor protecting against the induction of cell death by these drugs in stimulated primary splenocytes.



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Fig. 5. Induction of cell death by fluoropyrimidines in Ung-proficient and Ung-deficient cells. Cell death induced by 5-FU (A) and 5-FdUrd (B) in Ung+/+ (open squares) and Ung–/– (filled squares) activated primary splenocytes was measured by flow cytometry after 72 h of culture (n = 3). The percentage of living cells (PI negative) was used to calculate the survival relative to untreated controls. Similarly, the toxicity (as % survival) of 5-FU (C) after 72 h of culture of Ung+/+ (open squares) and Ung–/– (filled squares) MEFs was measured by the MTT assay and given relative to untreated control (n = 3).

 
Presence of the Ung enzyme did not influence toxicity of 5-FU in MEFs as measured by the MTT assay (Figure 5C) and the colony formation assay (data not shown). Similarly, the initial toxicity in response to 5-FdUrd was similar for both cell lines (data not shown). Thus, the induction of cell death by either drug does not depend on uracil excision by the Ung enzyme in these cell lines.


    Discussion
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 Abstract
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 Materials and methods
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 References
 
Previous estimates of steady-state levels of 10–15 uracil residues/diploid genome in human lymphocytes were based on measurements of the size of the dUTP pool (7). The availability of mice and cell lines deficient in the Ung enzyme has allowed us to analyse directly the level of uracil introduced into DNA in mammalian cells under normal growth conditions. Our results show that the level of uracil in the genome of wild-type mouse cells is below the limit of detection of the alkaline elution assay, i.e. <5 residues/108 bp (300 per diploid genome). It is interesting to note that in wild-type cells, the basal level of Fpg-sensitive sites (probably mostly 8-oxoG) is much higher than the level of UNG-sensitive sites, but that the increase of Fpg-sensitive sites in Ogg1–/– cells (37) is much smaller than the increase of uracil in Ung–/– cells, with the level of uracil in Ung–/– MEFs reaching ~3600 residues/genome (see Figure 1A).

Interestingly, the level of uracil in DNA in Ung–/– MEFs decreased by >50% after confluence was reached (Figure 2A). The clear correlation between the DNA uracil level and cell proliferation was confirmed using primary splenocytes from Ung–/– mice, where uracil levels increased >2-fold after stimulation with mitogens (Figure 2C). Smug1 contributes to the majority of the UDG activity in Ung–/– mouse cells and tissues (15) and appears, in contrast to Ung2, to be constitutively expressed throughout the cell cycle (12). It is therefore reasonable to assume that the repair capacity for uracil remains constant in cycling and non-cycling Ung–/– cells. We cannot formally exclude that the generation of uracil from cytosine by either hydrolytic or enzymatic deamination could be somewhat higher in actively dividing cells. However, we consider it unlikely that this would contribute more than the ‘established’ values of 100–500 uracils/cell/day (1), which is below the detection limit of our assays. Our data thus indicate that misincorporation during replication is a more abundant source of genomic uracil than cytosine deamination. In accordance with misincorporation being the major source of uracil in DNA, no elevation of the genomic uracil level was apparent in cells isolated from organs of Ung-deficient mice with very low turnover rates, such as mature spermatozoa. Support for this conclusion has recently come from genetic studies in yeast (38). The authors showed that inactivation of the Ung gene rescued the lethality of mutants deficient in AP-site repair, indicating that uracil in DNA was a major source of endogenous AP-sites. Furthermore, the failure of human TDG to rescue lethality suggested that the uracil residues originated from misincorporation of dUMP rather than from spontaneous deamination of cytosine.

In accordance with biochemical data (12), the presence of plateau of about 3600 uracil residues in the Ung–/– MEF would suggest that the back-up enzymes, Smug1 in particular, are able to remove incorporated uracil in the absence of Ung2, albeit less efficiently. Our results suggest that these constitutively expressed UDGs appear to be sufficient to prevent uracil accumulation under conditions of limited growth. However, as Smug1-induced repair would probably not be tightly coupled to replication (12), the time of residency of each misincorporated uracil residue in DNA would increase, thereby increasing the risk of inappropriate processing by DNA repair or damage-tolerance pathways.

The availability of Ung–/– and Ung+/+ cells also allowed us to analyse the causal relationship between uracil incorporation and subsequent excision, and the cytotoxicity of TS inhibitors, such as the fluoropyrimidines 5-FU and 5-FdUrd. Our data indicate that both 5-FU and 5-FdUrd induce uracil incorporation in the DNA of Ung–/– but not in wild-type MEFs (Figure 3A and C, respectively), showing that Ung2 is efficiently preventing uracil accumulation under these conditions. Both inhibitors caused a small increase in uracil levels in mitogen-stimulated wild-type splenocytes (Figure 4A and B), but we observed no dose-dependent increase of uracil in DNA in response to either drug in mitogen-stimulated Ung–/– splenocytes (Figure 4A and B). The apparent discrepancy between Ung–/– MEFs and splenocytes might be a result of several factors: a slower growth pattern combined with a lower fraction of actively growing cells in mitogen-activated cultures of primary splenocytes compared with MEF cultures, would leave a relatively small population of potential responders in the splenocyte cultures. A small increase in uracil content above the steady-state level in a minority of the population could, conceivably, be masked. Such an effect would be exacerbated in the Ung–/– splenocytes as the uracil levels in Ung–/– cells were close to the upper limit of detection, and probably no longer in the linear range for manual detection, even in the absence of TS inhibitors.

An intriguing finding here is that Ung–/– MEFs, but not wild-type cells, accumulate AP-sites as well as uracil in response to 5-FdUrd (Figure 3A). AP-sites have been shown to present a strong block to replication in yeast (39). Thus, the accumulation of AP-sites in Ung–/– MEFs could explain the stronger cytostatic effect of 5-FdUrd in Ung–/– MEFs (IC50 of 10 nM) compared with wild-type MEFs (IC50 of 200 nM) shown in Figure 3B. It was demonstrated recently that nuclear human UNG2 is part of a large complex, or complexes, also containing AP-endonuclease (APE1), DNA polymerase ß, XRCC1 and a DNA ligase, but not SMUG1 (40). This complex probably ensures proximity of APE1 to the abasic site, thus facilitating efficient incision and repair of this intermediate in wild-type cells. Our results suggest that the organization of repair steps is relaxed in the absence of an Ung2-associated complex, resulting in a less coordinated and less efficient repair with subsequent accumulation of abasic sites.

The lack of accumulation of AP-sites in Ung–/– MEFs in response to 5-FU in spite of significant uracil incorporation (Figure 3A) warrants an explanation because this suggests that uracil repair remains highly coordinated, albeit less efficient, in the absence of Ung2. Thus, the accumulation of repair intermediates in Ung–/– but not in wild-type MEFs, points to 5-FU incorporated into DNA (5-FUra) as a possible source of AP-sites and SSB in response to 5-FdUrd. Apart from UNG (12,41), both SMUG1 (12) and TDG (42) can remove 5-FUra when paired with adenine. MBD4 (43) and TDG (42) excise 5-FUra when base paired with guanine. Thus, Smug1 and Tdg would be able to remove newly incorporated 5-FUra residues in the first replication cycle, whereas Mbd4 and Tdg would be expected to remove 5-FUra from 5-FUra:G mismatches generated in the second round of DNA synthesis from misincorporation of dGMP opposite 5-FUra (see ref. 44 for review). The uncoupling of enzymatic steps of damage excision and AP-site incision in the absence of Ung2 is reminiscent of a state of imbalanced BER achieved through over-expression of DNA glycosylases. This has been shown to generate a strong mutator phenotype in yeast as error-prone translesion polymerases utilize AP-site containing DNA as template (45).

Moreover, the accumulation of repair intermediates in response to 5-FdUrd but not 5-FU is suggestive of a possibly important difference in the cytotoxic mechanisms of these two fluoropyrimidines: thymidine kinase is known to efficiently phosphorylate 5-FdUrd to 5-FdUMP, which is the major primary metabolite (46). 5-FdUMP inhibits TS but is also precursor for 5-FdUTP, which is a substrate for DNA polymerases. Inhibition of TS is likely to increase the dUTP/dTTP and 5-FdUTP/dTTP ratios explaining the incorporation of uracil, and most likely 5-FUra, into DNA. 5-FU as a prodrug is also metabolized to 5-FdUMP and 5-FdUTP, but is to a large extent converted to 5-FUTP that may in turn be incorporated into RNA by RNA polymerases (46; reviewed in ref. 20). As eluded to earlier, this may explain why, under the conditions used, 5-FdUrd has a stronger effect on proliferation in MEFs than 5-FU (Figure 3B and D), but, overall, our results are more consistent with depletion of dTTP pools as a major mechanism of action for 5-FdUrd, with incorporation of uracil (from dUTP) (36) or 5-FUra (from 5-FdUTP) playing minor roles. For 5-FU the mechanism may be more complex and may include toxicity from incorporation of the base analogue into RNA as discussed by Longley (20). Furthermore, it should be noted that the interconversions of fluoropyrimidines are complex, and 5-FdUrd is to some degree converted to 5-FU by thymidine phosphorylase. The contribution of different mechanisms is most likely cell-specific and dependent on the relative activities of a number of enzymes, only some of which are discussed here. In addition, a number of other proteins may have an effect on cell killing, or resistance, of cells treated with fluoropyrimidines. Our understanding of the mechanism of action of these drugs are likely to improve as a more complete picture of the molecular players involved is starting to emerge from recent microarray experiments measuring changes in mRNA expression levels after treatment (reviewed in ref. 20; 4749). In agreement with the present data, this approach has failed to identify uracil excision by Ung as a central modulator of TS inhibitor toxicity in mammalian cells. Although the present study does not offer an alternative mechanistic model for TS-inhibition cytotoxicity, it should serve to promote new research into this field as we provide evidence that cell death is activated equally well in Ung–/– MEFs that do accumulate uracil as well as in Ung+/+ MEFs that do not. This strongly suggests that accumulation of uracil in DNA is not required to initiate cell death, and that expression of the nuclear Ung enzyme has little influence on the overall toxicity of fluoropyrimidines in MEFs. Thus, our data would suggest that although the Ung enzyme is required to limit accumulation of uracil and repair intermediates in response to TS inhibition, it does not appear to have an effector function in induction of the cell death response.


    Notes
 
4 Present address: Hilde Nilsen, University of Oslo, The Biotechnology Centre of Oslo, PO Box 1125 Blindern, N-0317 Oslo, Norway Back

* Both authors contributed equally to this work. Back


    Acknowledgments
 
We thank Dr Tomas Lindahl for critical reading of the manuscript, Dr Deborah E.Barnes and Graham Daly for generously providing Ung knockout mice and cell lines. Financial support was from the Norwegian Cancer Society, The Research Council of Norway, The Cancer Fund at St Olav's Hospital (S.A., R.S., H.E.K.), Marie Curie Fellowships and Cancer Research UK (H.N.) and the Deutsche Forschungsgemeinschaft (Ep11/51 to B.E.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 31, 2004; revised October 27, 2004; accepted November 21, 2004.





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