The Effect of Methotrexate on Levels of dUTP in Animal Cells*

The amounts of intracellular nucleotides of dThd and dUrd were measured in cultured human lymphoblasts with and without treatment with methotrexate (plus hypoxanthine) to inhibit thymidylate synthetase. d'ITP fell from -40 pmol/106 cells (untreated) to -1 pmol/lO" cells with drug treatment. the dvTp/dT" 1 cells -0.2 cells methotrexate, that dUMP may be incorporated in substantial amounts into the DNA of the drug-treated cells. The magnitude of these effects on nucleotide pools suggests the possibility that the normal nucleolytic mechanism for removal of dUMP from DNA may play a part in the toxic effects on the cell that result from depression of thymidylate synthetase activity. the same as described above for lymphoblasts with the modZcations to increase sensitivity for detection of dUTP, including periodate treatment and purification of the dUrd (derived from dUTP) by paper chromatography before analysis by high pressure liquid chromatography (above). Specific activity of r3H]dTTP in the cell extract was determined as described for the lymphoblast cell l i e (above).

Inhibition of thymidylate synthetase is known to result not only in depression of intracellular dTTP (28-32), but increase in dUMP, as well (29, [32][33][34][35][36]. If the high intracellular concentrations of dUMP lead to sigmfkant increases in dUTP, this, together with the reduced dTTP, would be expected to favor greater incorporation of dUMP into DNA. Measurements of cellular dUTP pools have not been available previously, presumably because the levels are below limits of detection by methods ordinarily used.
In the studies described here, a sensitive procedure is used to determine dUTP levels in cells with and without inhibition of thymidylate synthetase with methotrexate. Combined with measurements of dTTP under the same conditions, the data allow predictions about relative rates of incorporation of dUMP and dTMP into DNA in normal and methotrexateinhibited cells. The results suggest a possible mechanism for some of the toxic effects of such drugs that is related to cellular levels of dUTP and dTTP.

MATERIALS AND METHODS
Cell Culture and Labeling-Unless noted otherwise, the experiments were carried out on a cultured line of human lymphoblasts (8866), which were grown in suspension in modified Eagle's medium as previously described (37). Hypoxanthine (25 PM) was added at the start of each experiment to avoid effects of methotrexate on purine synthesis; serine and glycine were present in the medium. All experiments were carried out in logarithmic phase of growth, at 7 to 9 X lo5 cells/ml. Cell doubling time was 16 h. Earle's salts (Flow) (5% COz) plus additions of NaHC03 (16.7 mM), ton, VT, were grown in Minimal Essential Medium (Eagle) with antibiotics (penicillin, streptomycin, nystatin), Na pyruvate (110 pg/ ml), vitamins, nonessential amino cells, L-glutamine, and fetal calf serum (10%). A roller bottle (1600 cm2 surface area) was seeded with 10' cells in 150 ml of the same medium. After 5 days the medium was replaced (100 m l ) and 18 h later (-90% confluency) the cells were labeled for 30 min with 0.1 p~ [6-3H]dUrd (20 Ci/mmol).
Measurement of Radioactivity in dUTP from Labeled Cells-Labeled lymphoblasts (2 to 10 X 10' cells total) were harvested by centrifuging (1600 X g) in a prewarmed centrifuge (30-37OC) for 40 S; with minimum delay, the medium was removed and the cell pellet suspended in 1 ml of 7.5% trichloroacetic acid containing 100 m o l of dUTP to serve as internal standard and marker for subsequent steps. The total time from removal of cells from culture vessel to addition of acid was 60 to 90 s. Trichloroacetic acid was extracted with noctylamine plus Freon (39); the aqueous phase was neutralized with Tris base, diluted to 100 ml with water, and applied to a column (1 ml bed volume) of DEAE-Sephadex A-25 (HCOa-) (Pharmacia). The column was washed with 20 ml of water and 20 ml of 0.02 M triethylammonium bicarbonate (pH 8.0) to remove salts and nucleosides; followed by 10 ml of 1 M triethylammonium bicarbonate to elute nucleotides. The sample was evaporated to dryness to remove triethylammonium bicarbonate, dissolved in 0.1 ml of water and c k~matographed (descending) 17 h on Whatman No. 17 paper in ammonium isobutyrate (isobutyric acid/water/concentrated NH4OH; 6 6 33:1, v/v/v). The dUTP region was identified with the aid of external markers, the internal dUTP marker, and radioactivity (chromatogram cut into 1-cm strips and counted in toluene scintillation fluid) in dTTP, which migrates ahead of dUTP; all three methods of locating dUTP in the paper chromatogram were needed because of imperfect correspondence in some instances between internal and external markers, and overlap of internal markers with UV-absorbing components in the sample. After removal of the scintillation fluid by washing with toluene and ethanol, the strips containing dUTP region then were eluted with water and successively chromatographed (and reeluted) two additional times, each for 72 hr, on Schleicher & Schuell 589 Orange Ribbon-C paper in ammonium isobutyrate. The sample was next treated with 50 pg of bacterial alkaline phosphatase (BAPC, Worthington) in a volume of 50 pl of Tris-HC1, pH 8.5, 10 mM MgCIz, for 3 h at 37°C. The phosphatase was precipitated with 5% trichloroacetic acid, and the acid was extracted as above. The sample was then analyzed for amount (At%) of dUrd and associated radioactivity by high pressure liquid chromatography on a reverse phase column (Lichrosorb RP-18, 4.6 X 250 mm, AlteX) using 20 mM potassium phosphate, pH 3.7, at 1.5 ml/min. Fractions (0.2 min) were collected directly into scintillation vials and counted in a Triton X-lOO/toluene scintillation mixture. The difference in retention time between UV detector and fraction collector was calibrated with labeled nucleoside. Recoveries of dUrd derived from the internal standard (dUTP) ranged between 10 and 4076, and this was used to correct the radioactivity in dUrd for losses during the procedure. Fractions with low radioactivity were counted a minimum of 60 min (6 X 10 min).
The procedure described above, carried out on untreated and methotrexate-treated lymphoblasts, was the source of most of the data presented in this report. Where noted, a modified procedure was used on untreated cells in an attempt to increase sensitivity for detection of dUTP. The modifications consisted of 1) oxidation of the extract with periodate (40) just prior to the DEAE-Sephadex step; and 2) further pdlcation, after dephosphorylation to dUrd, by paper chromatography in 1-butanol/NH,OH (see below) before the final analysis by high pressure liquid chromatography. The procedure was otherwise as described above.
Specific Activity of [3HJdUrd-labeled Nucleotide Pools-Specific activity of [3H]dUMP in methotrexate-treated cells was determined on acid extracts of [3H]dUrd-labeled drug-treated cells (50-ml culture volumes) prepared as in the preceding, except that prior to acid extraction, the cells were washed once with 0.15 M NaC1,0.02 M Tris.
Specifc activity of ['HIdTTP in untreated cells labeled with [3H]dUrd was determined using the same procedure as described for specific activity of [3H]dUMP in drug-treated cells. The choice of dUMP and dTTP in untreated and treated cells, respectively, was based on their relative abundance in each case, to facilitate measurement of amount ( A~M ) without interference from UV-absorbing contaminants. In several experiments specific activities of dUDP (methotrexate-treated cells), or dTDP and dTMP (untreated cells), were also determined and found not to differ significantly from the specific activities of dUMP and dTTP, respectively.
Analysis of Intracellular dTTP in Methotrexate-treated Cells-Unlabeled lymphoblasts, treated with methotrexate (6 h) as described above, were extracted with trichloroacetic acid containing 1 pmol of C3H]dTTP (60 Ci/mmol)/lO* cells as internal standard. dTTP was purified by chromatography in ammonium isobutyrate, then dephosphorylated and the dThd was purified by chromatography in 1butanol/NHaOH (see above). Losses were corrected for by the radioactivity of the internal standard, and dThd was quantified by a sensitive enzymatic assay in which the dThd was phosphorylated to dTMP with thymidine kinase and [y3*P]ATP. Samples containing 0.5 to 10 pmol of dThd were incubated 3 h in a mixture (20 p l ) containing 50 m~ Tris.HC1, pH 7.6, 5 mM MgCI2, 0.  Fractions containing dUMP were identified by external markers (indicated above) and the peak of radioactivity. B, nucleotide was eluted from dUMP-containing strips (A, bracket), converted to nucleoside with phosphatase, and chromatographed on paper in l-butanol/formic acid solvent system. After counting, the dUrd-containing strips (bracket) were eluted.  Fig. 1) was applied to a reverse phase column under conditions that resolve nucleosides, and the effluent monitored for UV (A250 and radioactivity ("Materials and Methods").
albumin, 10% glycerol, and Escherichia coli thymidine kinase (Fraction V) (42) sufficient to convert >95% dThd to dTMP. The mixture was chromatographed with dTMP and dThd markers on polyethyleneimine cellulose thin layer plates using 0.4 M LiCI. Paper chromatography in ammonium isobutyrate confirmed that the radioactive product was dTMP and not dUMP. 32P in dTMP gave the amount of dThd in the sample, easily detecting 0.2 pmol (Fig. 3). Conversion of the [3H]dThd to dTMP indicated completion of the reaction. Some samples showed non-linearity in the kinase reaction before, but not NHrOH. after, the purification by paper chromatography in 1-butanol/ Other Nucleotide Measurements-For untreated cells, the amounts of total acid-soluble radioactivity represented by dTTP, dTDP, and dTMP were determined on extracts from [3H]dUrd-labeled cells by paper chromatography in ammonium isobutyrate ( Fig.  4.4). These, together with the specific activity for dThd nucleotide (measured on d T T P see above), gave the amounts of each dThd nucleotide. The dUMP and dUDP regions of the chromatogram (Fig.  4A) were eluted, dephosphorylated, and chromatographed (as dUrd) on paper in l-butanol/NH40H to determine radioactivity in each; specific activity of dTTP was assumed to apply to dUrd nucleotides, as well, in untreated cells.
In r3H]dUrd-labeled methotrexate-treated cells, -98% of the acidsoluble radioactivity was in dUMP (Fig. 4B). Approximately 2% of the radioactivity was in the region of dUDP on the chromatogram (Fig. 4B) and when this was analyzed as described above for dUDP in untreated cells it was found that about half was in dUDP. The remaining -1% of radioactivity was present in a compound the identity of which is currently under investigation. The specific activity for [3H]dUMP in drug-treated cells (above) was used to convert radioactivities in dUMP and dUDP to picomoles, as described for the preceding.
Measurement of dUTP in Untreated NB Cells-At completion of labeling (see above), the medium was removed rapidly from the roller bottle and the cells (4.9 X los, determined as DNA) were covered immediately with 20 ml of cold trichloroacetic acid (10%) containing 50 m o l of dUTP. Total time from beginning removal of medium to complete immersion of cells in acid was -15 s. The remainder of the procedure was the same as described above for lymphoblasts with the modZcations to increase sensitivity for detection of dUTP, including periodate treatment and purification of the dUrd (derived from dUTP) by paper chromatography before analysis by high pressure liquid chromatography (above). Specific activity of r3H]dTTP in the cell extract was determined as described for the lymphoblast cell l i e (above).

Measurement of Intracellular dUTP
Cells not Treated with Drug-In the procedure that was used to measure dUTP, deoxyuridylate pools were labeled b y growing cells with [3H]dUrd. With the level of r3H]dUrd used here (0.1 p~) , in 30 min, untreated cells incorporated 7 to 13% of total label from the culture medium into DNA, and another 2.0 to 2.2% was in the acid-soluble fraction, almost entirely in the form of deoxythymidine nucleotides (Fig. 4A). With a  Thus, by 30 mi n, at which time cells were collected for extraction, the labeling conditions approached a steady state, and the specific activities of cellular nucleotides of dUrd and dThd were assumed to be very similar. Since the r3H]dUrd was diluted -20-fold by cellular pools (see specific activity, below), the perturbation of cellular nucleotide pools by labeled nucleotides in the medium is assumed to be negligible at these concentrations, paralleling observations on the effects of similar concentrations of dThd upon cellular pools of deoxythymidylate (43).
Using these labeling conditions, dUTP was isolated from the acid-soluble extract by successive paper chromatography (Fig. 5). A major function of the three paper chromatographic steps was to assure absence of dUDP, and to reduce contamination of the sample with the large amounts of radioactivity from dTTP, which migrates immediately ahead of dUTP (Fig.  5). The dUTP was converted to dUrd with phosphatase, and analyzed by high pressure liquid chromatography (Fig. 6). In spite of adequate recoveries of the d U T P internal standard (24 to 40%) ("Materials and Methods"), in three different experiments there was no radioactivity specifically associated with dUTP (Fig. 6). The identities of the radioactive components in the d U T P region of the paper chromatograms ( Fig.  5 and 6) are not known.
An experiment was also carried out with additional steps to improve sensitivity for detection of small amounts of dUTP. By including oxidation with periodate (to remove ribonucleotides) and an additional purification step (as dUrd) the remaining UV components were eliminated, but small amounts of the closely associated radioactive components remained. In some fractions, a very small amount of radioactivity appeared in the position of dUrd in the chromatogram (Fig. 7 ) . The specific activity for intracellular [3H]dTTP from cells labeled under the same conditions ranged from 537 to 648 cpm/pmol (three determinations), with an average of 590 ("Materials and Methods"). Using this value for the specific activity of dUTP, as well, the highest amount of radioactivity associated with dUTP ( Fig. 7 ) corresponds to 0.3 fmol/106 cells. However, considering the small amounts (<1/3 background) and the remaining radioactive contaminants in close proximity (Fig. 7 ) , the significance of this is uncertain.
Methotrexate-treated Cells-Cells were treated with methotrexate in the presence of exogenous sources of purine, serine, r3H]dUrd derived from r3wduI'P in untreated cells, using modified procedure. The procedure was carried out as described for Figs. 5 and 6 except that additional steps of periodate oxidation (prior to paper chromatography) and paper chromatography as [3H]dUrd (prior to high pressure liquid chromatography) were carried out to enhance sensitivity. One of the two final fractions is represented here.

dUTP in Methotrexate-inhibited Cells
and glycine to restrict the effects of the drug to inhibition of thymidylate synthetase. Here, the labeling period with [3H]-dUrd (2 h) was longer than for untreated cells, to assure equilibration with the expanded cellular pools of deoxyuridylate (29, [32][33][34][35][36]. Kinetic measurements of [3H]dUrd uptake into the acid-soluble fraction of drug-treated cells showed rapid initial uptake followed by slow linear rise for 4 h, with the value at 15 min 85% of the value at 2 h (not illustrated), indicating that labeling of deoxyuridylate pools approached steady state conditions at 2 h. Under these conditions, 0.015 to 0.02% of the total radioactive label in the medium was incorporated into DNA, and another 11 to 13% was present in the acid-soluble fraction from cells. Although there was a larger proportion of [3H]dUrd in the acid-soluble fraction (compared to untreated cells), dilution with the greatly expanded endogenous deoxyuridylate was 50-fold (see specific activity, below), again indicating an insignificant effect of the exogenous r3H]dUrd on cellular pool size.
The procedure for isolation of dUTP from the methotrexate-treated cells was the same as that described above for untreated cells. Here, the major radioactive component was dUMP (Fig. 4B), but this was removed with the first paper chromatogram, leaving dUDP as the major contaminant to be separated (Fig. 8, A and B), and this appeared to have been accomplished by the third paper chromatographic step (Fig.  8 0 .
In contrast to the results with untreated cells (above), samples from methotrexate-treated cells showed significant amounts of radioactivity associated with dUrd derived from dUTP (Fig. 9). The possibility that the radioactivity may have been from residual trailing contamination with dUDP (or an unidentified dUrd-containing compound located between dUDP and dUTP in isobutyrate chromatography) appears very unlikely, since: 1) the radioactivity in dUrd exceeds what was actually present in dUDP (Fig. 8 0 , even allowing for the lower efficiency for counting 3H-labeled nucleotide in paper (-one-third to one-sixth of the efficiency for Triton/toluene mixture); and 2) the ratio of radioactivity to A254 is essentially the same for the two fractions (Fig. 9, A and B), whereas it would be much larger for the fractions closer to dUDP (or the unknown compound) (Fig. 8, C, Ill if there had been a significant contribution of radioactivity due to trailing. The specific activity for [3H]dUMP in these cells ranged from 180 to 278 cpm/pmol (five experiments), with an average of 219. Using this for the specific activity of dUTP, and figures for recovery of purified dUTP from the internal standard (10 to 25%), the intracellular dUTP, for three different experiments, was 0.082, 0.222, and 0.264 pmol/106 cells.

Cellular Levels of Other dUrd and dThd Nucleotides
Availability of the specific activity for [3H]dTTP in untreated cells facilitated estimation of cellular pools for the other dUrd and dThd phosphates (all of which were assumed to have the same specific activity as dTTP) by simple assessment of the proportion of total acid-soluble counts represented by each ("Materials and Methods"). The specifk activity for dUMP in methotrexate-treated cells was used similarly to determine values for dUMP and dUDP. Because the specific activities of dThd and dUrd nucleotides were not the same in methotrexate-treated cells, dTTP was measured in these cells by an enzymatic assay for dThd using thymidine kinase and [y-32P]ATP, after isolation as dTTP and conversion to dThd with phosphatase ("Materials and Methods").
The results are summarized in Table I. dUMP was -0.8 pmol/106 cells without drug treatment and this rose -1400fold with methotrexate. dUDP also increased greatly with drug treatment (-300-fold). The level for dTTP in untreated cells was -40 pmol/106 cells, similar to previously reported values (32, 44,45), and with the methotrexate treatment this fell to -1 pmol/106 cells. As stated above, dUTP was not clearly detected in untreated cells (50.3 fmol/106 cells), but with methotrexate treatment dUTP had risen at least 700fold, to -0.2 pmol/106 cells. Thus, with drug treatment the ratio of dUTP/dTTP had risen from 510-5 to -0.2.  (Fig. 8C), were converted to nucleoside and analyzed by recovery of internal standard (A254) and radioactivity in r3H]dUTP from [3H]dUrd-labeled cells, as in Fig. 6. A represents Fraction I, and B, Fraction 11.

Intracellular dThd and dUrd nucleotides
The procedures for the determinations are given under "Materials and Methods." Each represents the mean for three or more separate experiments except for dUDP, which was measured twice. Range is given in parentheses.

dUTP in NB Cells
To test whether the very low level of dUTP in untreated lymphoblasts is a peculiarity of that cell line, another cell line, SV-40-transformed human newborn kidney cells, was analyzed. Even with the modified procedure of greater sensitivity, no dUTP was detected in these cells (50.6 fmol/106 cells).
The level of dTTP in the NB cells was 60 pmol/106 cells.

DISCUSSION
Inhibition of thymidylate synthetase in cultured lymphoid cells, resulting from inactivation of dihydrofolate reductase by methotrexate, caused a fall in dTTP to about one-fortieth of its normal value. Reduction in dTTP pools with inhibition of thymidylate synthetase, measured by the DNA polymerase assay, has been reported several times previously, although in most cases the degree of reduction has not been as great as found here (28)(29)(30)(31)(32). The rise in dUMP accompanying the fall in dTTP has also been described before (29,(32)(33)(34)(35)(36); most of the earlier reports showed a much smaller effect on dUMP, e.g. up to a maximum of 13-fold increase (29,32,33,35,36), in contrast to the >lo3-fold increase found in the present study, although in one of the most recent studies, there was 300-fold increase in dUMP (34). It should be noted that the dUMP levels in untreated cells found in some of the earlier studies, which employed a thymidylate synthetase assay procedure (29,33,35,36), were much higher (-lo2 to lo3 times) than found here. Whether the differences are due to cell differences, type or amount of drug used, or the assay method, is not clear at this time. In more recent reports, the levels of dUMP determined both by the method used previously (12 to 15 pmol/lOfi cells) (32) and a modified form of the procedure (2 to 5 pmol/106 cells) (34) were closer to the values found in the current study.
Available evidence indicates that the increase in dUMP that occurs with inhibition of thymidylate synthetase is largely a result of "de-inhibition" of dCMP deaminase due to the fall in dTTP (32,(46)(47)(48). Additional contributions to the rise in dUMP come from the non-utilization by thymidylate synthetase, and increases in activity of ribonucleoside diphosphate reductase (converting UDP to dUDP, and CDP to dCDP) (32,(48)(49)(50), and thymidine kinase (32,46, 51) (reducing excretion as dUrd), the enzyme activations in each case a result of the fall in dTTP.
The high dUMP level is not a t present known to have physiological consequences, although one effect may be to exert some control upon its own level. It has been shown that dUMP inhibits the activity of dCMP deaminase ( I C L = 0.21 mM) (32). With the dUMP concentrations reported here in drug-treated cells, inhibition of dCMP deaminase may play a part in preventing even further increase in dUMP, although this mechanism may be of little significance for lower levels of dUMP (29,32,33,35,36). An additional effect of the high levels of dUMP may be to limit rate of utilization of exogenous thymidine by competing at the level of dTMP kinase. This may account, in part, for the poor incorporation of ['HIdThd in methotrexate-treated cells.' It has also been suggested that reduced incorporation of r3H]dThd into DNA under similar circumstances may result from competition of dThd with increased dUrd at the thymidine kinase step (32), but, in contrast to a previous report (32), there was little or no evidence for intracellular accumulation of dUrd in the experiments reported here.
It is shown in the present study that the increase in dUMP in methotrexate-treated cells is associated with a marked increase in dUTP, as well, presumably via dTMP kinase and nucleoside diphosphate kinase. It was not known before these results whether significant accumulation of dUTP would ensue in the face of the highly effective mechanism opposing it. The enzyme dUTPase, which hydrolyzes dUTP to dUMP and PPi, functions as a major factor in preventing accumulation of dUTP (2-7, 16, 17,20,21,25-26). Intracellular concentrations of dUTP normally are extremely low primarily because of this mechanism and the present analyses give a figure, not previously available, for an upper limit in untreated cells. The figure may be compared with indirect estimates of -0.5 p~ for E . coli (7) and 1/200 of dTTP concentration (18). Using the estimate of 1 p1 for intracellular volume of lo6 cells, the dUTP concentration in lymphoid cells is 50.3 nM. It is possible that a higher level of dUTP in E. coli compared to the level in animal cells reflects the utilization by prokaryotes of the dUTPase reaction as the major source of dUMP (52, 53). In prokaryotes, dCTP is converted to dUTP by dCTP deaminase, which is absent in animal cells.
Although not detected in untreated cells, dUTP was easily ' M. Goulian 6, 24, 54)). With methotrexate treatment, dUTP evidently rises until it reaches a level at which the rate of hydrolysis by dUTPase equals the greatly increased rate of formation of dUTP under these circumstances.
Since DNA polymerases utilize dUTP (8, 9), the approach of dUTP concentration to that of dTTP in the methotrexatetreated cell implies that significant incorporation of dUMP into DNA must occur in the course of the residual DNA synthesis in these cells. The presence of dUMP in DNA activates a mechanism for its removal which is initiated by removal of the base Ura by the enzyme, Ura-DNA glycosylase (7, 12-15). The resulting apyrimidinic site is incised by an apurinic/apyrimidinic endonuclease, followed by excision and repair (13, 20, 55, 56).
It is commonly assumed that the principal mechanism for cytotoxicity in methotrexate-treated cells is a direct result of levels of dTTP being inadequate to sustain synthesis. To what extent a fall in dTTP of the magnitude observed here would, by itself, affect the rate of DNA synthesis, with or without causing cell death, is not at present known.
The current studies point to a process that results both directly and indirectly from reduced dTTP concentration, and which may also have toxic consequences for the cell. In the course of gap fiiing for excision/repair at the site of dUMP insertion (see above), there is a significant probability of reinsertion of dUMP, resulting in re-initiation of the process of Ura removal/incision/excision/repair. This probability is a function of repair patch size and ratio of concentrations, dUTP/dTTP. As the concentrations of dUTP and dTTP approach each other, a point may be reached at which the rate of dUMP incorporation into DNA exceeds the net rate of dUMP removal, thus leading to cyclic repetition of the insertion/removal process, without gap closure and possibly even progressive net gap widening. Even if this point is not reached, the active process of incorporation/removal may result in double strand interruptions (especially if both strands are involved) or other irreversible alteration(s) in DNA structure.
In experiments reported elsewhere (57), it is shown that dUMP is present in DNA of cells treated with methotrexate, thus fulfiiing the prediction that follows from the measurements of dUTP and dTTP pools presented here. In addition, the dUMP in DNA increases with inhibition of Ura-DNA glycosylase (with Ura), and disappears rapidly if dThd is added to the culture, reflecting the active process of removal that accompanies dUMP incorporation (57).
In vitro experiments illustrate the fragmentation of DNA that may result from dUMP incorporation/removal at ratios of dUTP to dTTP of 1:lO or greater (24)(25)(26)(27). In one of the in vitro systems the fragmentation is irreversible, i.e. maturation to high molecular weight DNA does not ensue with removal of dUTP (24, 27). Although some dut or sof(dUTPase-defective) mutants of E. coli are able to grow essentially normally in spite of the greater fragmentation of newly synthesized DNA that results from the (presumed) higher levels of dUTP (16, 17, 58), strains with the most severe defect in dUTPase do show a growth defect (58).
Fragmentation of DNA has been observed previously in association with "thymine-less" states, both in thymine-requiring prokaryote mutants (59-64) and drug-treated animal cells (65-67). The possibility of a relationship of fragmentation to cytotoxicity is supported by the observation (in a prokaryote) that both are reduced by a defect in Ura-DNA glycosylase (22). However, additional information will be needed to establish what, if any, role is played by dUMP incorporation/ removal in the cytotoxicity that results from inhibition of thymidylate synthetase.