An antifolate-induced lesion in newly synthesized DNA

AN ANTIFOLATE-INDUCED IN NEWLY SYNTHESIZED

LESION DNA

W. DAVID SEDW1CK and JOHN LASZLO Departments of Medicine, and Microbiology-Immunology, Duke Medical Center. Durham, North Carolina, 27710

IN1"RODUCTION

Many of the earliest effects of antifolate toxicity result in perturbation of de n o v o nucleotide biosynthesis. Antifolate inhibition of dihydrofolate reductase (DH FR) and consequent tetrahydrofolate depletion have immediate impact on pyrimidine pools. Thymidine (TdR) pool sizes decrease (I), deoxycytidine pools typically increase (2), and deoxyuridine monophosphate (dU M P) pool sizes also increase in some cells (3). Since several biochemical pathways respond to tetrahydrofolate depletion, the relative toxicity of antifolates for different cells, or for the same cells under different conditions, may be determined by very complex biochemical interactions (4-6). The ability of cells to regulate induced dUMP levels, for example, may differentially, alter the metabolic consequences of antifolate action in the presence of exogenous nucleosides (4, 7). These differences in response to antifolates can be regulated via enzymes involved in de n o v o nucleotide biosynthesis such as dihydrofolate reductase (8, 9), deoxycytidylate deaminase (4, 10), and ribonucleotide reductase (4, 6, I I), or through differences in enzymes of nucleotide salvage pathways (4, 12). Recently.renewed interest in these metabolic interactions that were documented over 20 years ago (13) has led to in vivo studies in both humans (14) and animals (15, 16) that have shown that methotrexate with TdR infusion may facilitate antifolate regimens by decreasing toxicity and increasing selectivity of the antitumor response. The study described below was directed toward elucidation of the role of exogenous deoxyuridine (UdR) in modulation of the expression of antifolate toxicity produced in cultured cells. This paper also describes an abnormality caused by antifolate-induced d U M P accumulation which has direct and specific effects on DNA synthesis. The studies (17) show that antifolates: (a) promote misincorporation of dUMP into DNA, and (b) can lead to specific inhibition of high molecular weight DNA synthesis with simultaneous accumulation of low molecular weight DNA as measured by alkaline sucrose gradients. We will present the background to elucidation of this lesion, with specific emphasis on the effects 295

296

w. DAVID SEDWICK AND JOHN LASZLO

of the lipid-soluble antifolate, 2,4-diamino-5-{3',4'-dichlorophenyl]-6-methylpyrimidine, (DDMP), on DNA synthesis. MATERIALS AND METHODS

Cell Culture Maintenance WIL-2, the human lymphoblastoid cell line utilized in this study, was obtained and maintained as described previously (18). Prior to each experiment the cells were incubated for one hr in fresh MEM supplemented with 10% fetal calf serum (dialyzed against three changes of 100 vol of 0.15 M NaCI for a period of 48 hr, or as obtained directly from Baltimore Biologicals, Inc.). The cell suspensions were then centrifuged at 2000 r.p.m, for 5 min and resuspended in fresh MEM growth medium.

Sample Preparation For Nucleotide Pool Analysis Cells were incubated in fresh growth media for 15-60 min prior to addition of 3H Ud R, 24.2 mCi/mmol. Cells were then harvested at the indicated times by centrifugation for 30 sec in an Eppendorf Microfuge at 10,000 r.p.m., which immediately removed the cells from the labeling medium. The supernatant was then discarded and the pellet suspended in 60% methanol at -20 ° C. Extraction of nucleosides and nucleotides was carried out for at least 24 hr before further processing of the samples.

Nucleotide Pool Analysis by Cellulose Thin Layer Chromatography The methanol nucleotide pool extracts described above were lyophilized to dryness and resuspended in 30 tal of 10% isopropanol, 10% acetic acid, and 80% water. DNA nucleotides (0.01 N) were used as standards for chromatography. Cellulose plastic sheets (Kodak) impregnated with fluorescent dye indicator were divided into ten numbered channels. Aliquots of each sample were spotted at the origin, positioned at 2 cm from the bottom of the plate for each channel. Aliquots of standard solutions were spotted together with, or beside, each sample, and the position of migration was visualized by irradiating the plates with short wave ultraviolet light. The solvent system utilized for the nucleotide separations was isobutyric acid: NH4OH: H20 (63:1:33). Thin layer chromatography plates were developed for approximately 4 hr (or until the solvent had moved 17 cm from the bottom plate), and then air dried overnight. The dried sheets were cut into strips following the demarcation of each channel, the strips were cut further into 0.5 cm sections, and placed in scintillation vials for counting. The position of the radioactive nucleotides was then recorded relative to the reference markers in the same a n d / o r adjacent channels.

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297

Alkaline Sucrose Centrifugation of DNA The procedure used for alkaline sucrose centrifugation of D N A was modified from Perlman et al. (19). Extracts were prepared by directly injecting the labeled samples into 60% methanol at -20 ° C, followed by incubation at this temperature for at least 24 hr before further processing. This procedure allows both soluble pool and DNA analysis to be carried out on the same samples. Cell pellets were prepared for gradient analysis by resuspending them in 0.3 M N a O H , 2% SDS solution and extracting them for three hr at room temperature on a Fisher (Roto Rack) rotator at 4 r.p.m. Alkaline sucrose centrifugation was performed in Beckman SW41 polypropylene centrifuge tubes on gradients of 10-30% (w/v), alkaline sucrose containing 0.7 M NaCI, 0.3 M N a O H , 10 mM Tris, and I mM EDTA. Alkaline extracts of the cell pellets were poured onto the gradients and centrifuged for 18 hr at 38,000 r.p.m, at 14°C. The gradients were fractionated into 0.3 ml aliquots. One hundred /~l of each fraction was pipetted onto W h a t m a n 3 mm paper discs which were processed and counted as previously described (20). Samples were also analyzed for incorporated counts by adsorbing aliquots of the fractions on D E A E c h r o m a t o g r a p h y paper (21). BSA, which sediments identically in alkaline and neutral sucrose gradients, was used as an internal centrifugation standard.

RESULTS

AND DISCUSSION

Studies on the Effect of Exogenous UdR on Antifolate-lnduced Toxicit.v These studies started several years ago when we explored the role of Ud R in promoting or alleviating toxicity in antifolate-treated cells. Although several consequences of adding UdR to cultures seemed possible at that time, the working hypothesis of the initial experiments was that a "leaky" block of T M P biosynthesis might cause more efficient depletion of the tetrahydrofolate pool. The rationale for this hypothesis was that exogenous UdR might be expected to increase the size of the d U M P pool, thereby enhancing synthesis of T M P and more rapid utilization of methylenetetrahydrofolate cofactor. Figure I, panels A and B, shows one experiment designed to test this hypothesis. Cells were completely inhibited for growth by I /~M MTX. Dead cells, as measured by trypan blue staining, also increased at equal rates in MTX-treated cultures in the presence or absence of UdR, Panel B. Moreover, MTX ( I tzM) maintained a blockade of UdR incorporation for at least 29 hr in the presence or absence of exogenous cold Ud R, Panel C. An identical level of isotopic dilution of 3H-UdR was also observed throughout the time course of the experiment when exogenous unlabeled UdR was added to the culture, showing that the concentration of UdR in the cells and culture medium did not change significantly over the 29 hr of the experiment, Panel C.

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F u r t h e r m o r e b o t h in the p r e s e n c e a n d a b s e n c e o f a d d e d U d R , the i n h i b i t i o n o f U d R i n c o r p o r a t i o n i n t o D N A r e m a i n e d c o n s t a n t w i t h time. W e c o n c l u d e d t h a t u n d e r these c o n d i t i o n s a d d i t i o n o f e x o g e n o u s U d R did not lead to a d i s c e r n a b l e i n c r e a s e in t o x i c i t y , e i t h e r in t e r m s o f cell s u r v i v a l o r UdR incorporation into DNA. T h e r e f o r e , we n e x t c h a r a c t e r i z e d the m e t a b o l i c r e s p o n s e o f cells to M T X a n d D D M P at v a r i o u s c o n c e n t r a t i o n s o f e x o g e n o u s U d R a n d T d R . F i g u r e 2 s h o w s t h a t at c o n c e n t r a t i o n s o f U d R r a n g i n g f r o m 0.1 to 12.5 ~M, the a p p a r e n t rate o f U d R i n c o r p o r a t i o n into D N A i n c r e a s e d p r o p o r t i o n a l l y in

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FIG. 2. Inhibition of deoxyuridine incorporation into D N A as a function of exogenous UdR concentration. WI L-2 cells (2.5 X I0~.' ml) were prcincubated for I hr in M EM containing0.005 ~,t Hepes buffer, pH 7.0 and I 0 eli dialyzed fetal calf serum. Cell suspension (0.2 m)) was lhen added to 0.3 ml of ~H-UdR and cold UdR to achieve the indicated UdR concentrations. DDM P ( I .uM) was added to drug-treated samples. After incubating for the indicated time periods 0.25 ml samples were removed, diluted with ice cold phosphate buffered saline, and processed as previously described. Regression lines plotted in panels A and B were calculated Io fil wilh a correlation coefficient of >.98 for all curves. Panel A No D D M P 0.1 ,M UdR (A).0.15,M UdR (n). or 0.4 pM UdR (O)" and I pM D D M P 0.1 pM UdR (",), 0.15 pM UdR IlI). or (~.4 ,M UdR ('): Panel B No D D M P -- 2/~M UdR ( I ) . and 12.5 ~M UdR to): 5 , M D D M P 2pM UdR and 12.5/~M UdR. The reaction velocities calculated from the data in Panels A and !] were then used to determine percent inhibition relative to control at each UdR concentration. Panel C.

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both drug-treated and untreated cells so that the percent of inhibition relative to control cells remained constant over the entire range of UdR concentrations. The apparent rate of UdR incorporation calculated from the reaction velocities in Figure 2 also increased proportionally with exogenous UdR concentration through 2 taM UdR. It was also of interest to determine whether restoration and augmentation of intracellular TdR pools would have an effect on antifolate-induced methylene tetrahydrofolate depletion-dependent inhibition of UdR incorporation into DNA. Therefore, we also characterized the competitive inhibition of TdR and UdR for each others incorporation into DNA of drugtreated cells, Figure 3, [Kmapp for T d R incorporation into DNA = 0.35 (not shown) and Kmapp for UdR incorporation into DNA = 1.25; K, = .06 taM for T d R vs UdR and K, = 2.2 taM for UdR vs TdR (not shown)]. Despite competitively inhibiting UdR incorporation into DNA, TdR did not affect D D M P or M T X inhibition of UdR incorporation into DNA, expressed as percent of inhibition relative to control cultures at each TdR concentration (0.1 to 1.0 taM), Figure 4.

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( TDR ) UM FIG. 4. Effect of TdR on inhibition of UdR incorporation by DDM P and MTX. 2.5 × 10~WI L-2 cells/ml were suspended and incubated as described in Figure 2. ~H-UdR (0.1 #M) was then incubated with the indicated concentrations of TdR in the presence of 0.5 #M MTX and I ~aM DDMP and the percent inhibition relative to control cultures was determined. The insert graph shows the data from which the percent inhibition values were calculated. D D M P establishes a steady-state of inhibition of UdR incorporation into D N A within 5 sec of its addition to W I L - 2 cells (23). In this regard D D M P is significantly different f r o m M T X which, because of its dependence on active transport for entry into the cell, requires a prolonged pretreatment period before a steady-state of inhibition can be established. This makes D D M P a much more attractive drug than M T X f o r study of metabolic perturbation of nucleotide biosynthesis and D N A synthesis. Moreover, drug concentration dependent steady-states of inhibition of U d R incorporation could be maintained for extended periods of time with D D M P . On the other hand, concentrations of M T X f r o m 0.05 to 5/~M established approximately the same level of inhibition in W I L - 2 cells, by virtue of the Concentrative ability of the active transport system for this drug, so that experiments with M T X could not easily be conducted at different steady-state inhibition levels.

Metabolism o f 3H-UdR in DDMP-Treated Cells O u r previous studies (23) also showed that the apparent size of the acidsoluble pools of intracellular Ud R synthetic metabolites increased with time in D D M P-treated cells to levels far in excess of those observed at steady-state in drug-free cells. The acid-soluble product(s) o f ~ H - U d R were unidentified in these experiments (23), however, so that it became important to clarify the

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intracellular fate of exogenous UdR. Figure 5 shows that exogenously a d d e d U d R led t o a n i n t r a c e l l u l a r b u i l d - u p o f d U M P , a n d a n a l y s i s f u r t h e r s u g g e s t e d t h a t d U T P m i g h t b e a d e t e c t a b l e p r e c u r s o r f o r D N A s y n t h e s i s in DDMP-treated cells (17). T h e p r e s e n c e o f d e t e c t a b l e levels o f d U T P w a s u n e x p e c t e d in t h a t e u k a r y o t i c cells w e r e k n o w n t o c o n t a i n a v e r y a c t i v e

ANTIFOLATE-INDUCED DNA SYNTHESIS LESION

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dUTPase (24, 25). Further experiments demonstrated that this expected specific phosphatase activity did not prevent accumulation o f d U T P , and that this nucleotide, not TTP, became the major detectable DNA substrate in DDMP-inhibited cells when UdR was added as a labeled exogenous precursor (17). Moreover, d U M P was found to represent >90% of the labeled nucleotide in the DNA of drug-treated cells, proving unambiguously that antifolates can promote misincorporation of UdR into cellular DNA. This result was consistent with the recent observations of Goulian et al. with MTXtreated lymphoblastoid ceils (26). Direct incorporation of d U M P into DNA in the presence of D D M P was also consistent with the dependence of UdR on the concentration of exogenous nucleoside. It is still noteworthy, however, that the relationship between the observed percentage of inhibition of UdR incorporation into DNA by 'antifolates also did not vary with changes in exogenous concentrations of UdR or by competitive interaction of UdR with TdR. This occurred even though UdR entered DNA as d U M P in the inhibited cell, whereas in the uninhibited cell, it was incorporated via T T P as TMP. Significantly, therefore, although UdR was incorporated into DNA as different nucleotides, the incorporation of UdR was rate limited proportionally by exogenous UdR in drug-free and DDMP-treated cells.

D N A Synthesis as Anah.zed by UdR and TdR Incorporation in D D M P Treated Cells Our latest studies have explored the effects of D D M P on progression of DNA synthesis in WIL-2 cells. In mutant bacteria (27, 28) and in permeabilized eukaryotic cells or extracts (29, 30) synthesis of small "Okazaki" fragments has been observed to occur as a consequence of d U M P misincorporation into DNA, and the response of the N-glycosylasedependent repair system to this lesion. DDM P-treated cells also appeared to contain small fragments of DNA, Figure 6, but the low level of incorporation in drug-treated cells made it difficult to unambiguously connect the presence of these fragments with d U M P misincorporation. We have also shown, however, that cells treated with DDM P alone may be blocked somewhere in progression of DNA synthesis, or alternatively may fragment their high molecular weight DNA. Detection of this lesion, with TdR as a labeled precursor in the absence of exogenous UdR, was only possible in experiments involving relatively long pulses of around 5 min (17). In these experiments DNA fragments of intermediate size appeared to accumulate that may be analogous to the 82S fragments reported by Fridland in MTX-treated cells labeled with deoxycytidine (3 I). The most pronounced DNA synthetic lesion, however, was evident when ~H-UdR was utilized as a substrate. Under conditions where D D M P

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s t r i n g e n t l y b l o c k e d i n c o r p o r a t i o n o f 3 H - U d R i n t o D N A as T M P , h : g h m o l e c u l a r w e i g h t D N A a c c u m u l a t i o n in d r u g - t r e a t e d cells was i n h i b i t e d , w h i l e l o w m o l e c u l a r w e i g h t D N A levels i n c r e a s e d w i t h t i m e , F i g u r e 7. T h e r a t i o o f l a b e l i n g o f high a n d l o w m o l e c u l a r w e i g h t D N A in c o n t r o l a n d D D M P - t r e a t e d cells at 40, 80 a n d 180 sec is s h o w n .

ANTIFOLATE-INDUCED DNA SYNTHESIS LESION

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SECONDS FIG. 7. Preferential incorporation of ~H-UdR into small DNA fragments in the presence of DDMP. WIL-2 cells, 3.7 × l07 cells/ml, were pulse labeled with 100 ~Ci (413 nmol) of ~H-UdR for 40, 80, and 180 sec, after no drug treatment and after pretreatment with 5/.tM DDMP for 15 min. At the end of the indicated pulse labeling periods cells were harvested, extracted with SDS and analyzed by alkaline sucrose gradient centrifugation as described in Methods, except that the precipitated cells from methanol extractions were subsequently washed 4 times with 60% methanol to remove unincorporated nucleoside from the cell samples before cell disruption and centrifugation. The sucrose gradient profiles of cells labeled with UdR in the absence of drug(O) and in the presence of DDM P (o) for 40, 80 and 120 see were reduced to counts incorporated into DNA of greater than 4S, and 4S and less. This was accomplished by summing the counts of each gradient from fractions I 23 and from fractions 24-33, respectively. The ratio of high and low molecular weight DNA in control vs. DDMP treated cells was calculated and plotted for each pulse labeling interval

Conclusions Cells treated with D D M P are i m p a i r e d in p r o d u c t i o n o f high m o l e c u l a r weight D N A , a n d a c c u m u l a t e small D N A . This o b s e r v a t i o n m a y be a c o n s e q u e n c e of r e p a i r - i n d u c e d f r a g m e n t a t i o n of newly synthesized D N A or an unspecified b l o c k to the p r o g r e s s i o n o f synthesis o f high m o l e c u l a r weight D N A . It is not k n o w n w h e t h e r the a c c u m u l a t i o n o f low m o l e c u l a r weight D N A represents c o n t i n u i n g initiation of new D N A replication forks that are greatly inhibited in their synthesis, or slowed p r o g r e s s i o n to larger D N A f r a g m e n t sizes in the inhibited cells. U d R , however, is i n c o r p o r a t e d into d e m o n s t r a b l y a b n o r m a l D N A in the presence of antifolates as an early d e t e c t a b l e m e t a b o l i c lesion. This D N A is not only m u c h smaller in average size t h a n is the D N A of d r u g - f r e e cultures, but also c o n t a i n s d U M P residues t h a t are p r e s u m a b l y i n c o r p o r a t e d in place of T M P . A l t h o u g h the c y t o t o x i c i m p a c t o f this lesion is u n k n o w n at this time, d o c u m e n t a t i o n of its existence in intact cells has i m p o r t a n t r a m i f i c a t i o n s for the possible toxic potential of d U M P a c c u m u l a t i o n b e y o n d the effects on T d R b i o s y n t h e s i s a n d t e t r a h y d r o folate d e p l e t i o n c o n s i d e r e d b y others (4, 6, 7). O u r earlier studies s h o w e d that D D M P a l o n e did not inhibit D N A synthesis, as m e a s u r e d by 32PO 4 i n c o r p o r a t i o n into D N A , until a b o u t I hr

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after drug addition to WIL-2 cells (32). Although the effect of a combination of UdR and D D M P on cellular synthesis rates was not studied in WIL-2 cells, it was clear that the rate of apparent incorporation of UdR into DNA was proportionally regulated by exogenous UdR concentrations. Moreover, neither the concentrations of UdR nor the competing nucleoside, TdR, affected the relative inhibition caused by D D M P over a broad range of exogenous nucleoside concentrations. However, the rate limiting step for Ud R incorporation under these conditions remains to be determined. Finally, our results showed that 5 /~M D D M P created a stringent block to T M P biosynthesis in WlL-2 cells. It follows from these results that the degree of tetrahydrofolate depletion caused by DDM P under these conditions would not be influenced by exogenous nucleoside concentrations nor by extensive shifts in nucleoside pool sizes of d U M P and TdR nucleotides. SUMMARY

Antifolates cause cells to accumulate exogenous UdR as d U M P in drugtreated cells. This accumulation of intracellular d U M P has no effect on inhibition of UdR incorporation into DNA. On the other hand, d U M P accumulation does result in detectable d UTP being synthesized and utilized as substrate for DNA synthesis. Although in the presence of metoprine (DDMP), d U M P is incorporated into DNA instead of TMP, the natural product of UdR substrate in the absence of the drug, DNA synthesis is equally rate limited by exogenous UdR. However, when analyzed on alkaline sucrose gradients, DNA which has incorporated exogenous UdR in the presence of antifolate sediments at 4S or less. This newly synthesized DNA accumulates preferentially while either synthesis of larger DNA is blocked, or high molecular weight DNA is preferentially broken down as a result of DNA repair. These observations further increase the metabolic complexity of pathways that must be considered as underlying the differential toxicity of antifolates. ACKNOWLEDGEMENTS

The authors appreciate excellent technical assistance from Messers Oliver E. Brown, Marc Kutler, and Thomas Frazer. Research support was provided by Grants from the Department of Health, National Cancer Institute (CA 0880 and CA 11265). REFERENCES M. H. N. T A T T E R S A L L , R. C. J A C K S O N . S. T. M. J A C K S O N and K. R. H A R R A P , Factors determining cell sensitivity to methotrexate: studies of folate and deoxyribonucleotide triphosphate pools in five m a m m a l i a n cell lines. Europ. J. Cancer 100 819 826 (1974).

ANTIFOLATE-INDUCED DNA SYNTHESIS LESION 2.

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