ICANCER RESEARCH 52, 533-539, February 1, 1992]

Cellular Elimination of 2',2'-Difluorodeoxycytidine 5'-Triphosphate:

A Mechanism of Self-Potentiation1

Volker Heinemann,2 Y¡-ZhengXu, Sherri Chubb, Alina Sen, Larry W. Hertel, Gerald B. Grindey,and William Plunkett1

Department of Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [V. H., Y. X., S. C., A. S., W. P.], and Lilly ResearchLaboratories, Indianapolis, Indiana 46285 (L. W. H., G. B. G.]

ABSTRACT

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidineanalogue which, after phosphorylation to the 5'-di- and 5'-triphosphate

(dFdCTP), induces inhibition of DNA synthesis and cell death. Weexamined the values for elimination kinetics of cellular dFdCTP andfound they were dependent on cellular concentration after incubation ofCCRF-CEM cells with dFdC and washing into drug-free medium. Whenthe drug was washed out at low cellular dFdCTP levels (<50 n\\),dFdCTP elimination was linear (t,: = 3.3 h), but it became biphasic atintracellular dFdCTP levels >100 JIM. Although the initial eliminationrate was similar at all concentrations, at higher concentrations theterminal elimination rate increased with increasing cellular dFdCTPconcentration, with a nearly complete inhibition of dFdCTP eliminationat 300 pM. The deamination product 2',2'-difluorodeoxyuridine was the

predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the majorexcretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydro-deoxyuridine transformed the monophasic dFdCTP degradation seen atlow dFdCTP levels into a biphasic process, whereas the deoxycytidinedeaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTPelimination. An in situ assay indicated that dCMP deaminase activitywas inhibited in whole cells, an action that was associated with adecreased dCTP:dTTP value. In addition, dFdCTP inhibited partiallypurified dCMP deaminase with a 50% inhibitory concentration of 0.46m\i. We conclude that dFdC-induced inhibition of dCMP deaminaseresulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

INTRODUCTION

dFdC4 (Gemcitabine) is a deoxycytidine analogue in whichgeminai fluorines replace both hydrogens of the 2' carbon atom

(1). An unusually broad spectrum of activity in murine tumors(2) and human tumor xenografts (3) provided impetus forevaluation of its anticancer activity in clinical trials (4-6). dFdCmust be phosphorylated by deoxycytidine kinase to exhibitcytotoxic and therapeutic activities (7,8). Its major intracellularmetabolite is dFdCTP, although it remains in a constant ratiowith the lesser metabolites, dFdCMP and dFdCDP (7).

DNA synthesis is specifically inhibited by dFdC by severalseparate mechanisms, (a) dFdCTP competes with dCTP as aweak inhibitor of mammalian DNA polymerase (9). Neverthe-

Received 8/21/91; accepted 11/13/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grant CH-130 from the American Cancer Society and

Grant CA28596 from the National Cancer Institute, Department of Health andHuman Services.

: Present address: Department of Internal Medicine. Hematology/Oncology,

Klinikum Gro¡ihadcrn,University of Munich, Munich. Germany.3To whom requests for reprints should be addressed.4 The abbreviations used are: dFdC, 2'.2'-difluorodeoxycytidine;CEM, CCRF-

CEM lymphoid cells; dFdCMP, dFdCDP, and dFdCTP, the 5'-mono-, dì-,andtriphosphates of dFdC; dFdU and dFdUMP, 2'.2'-difluorodeoxyuridinc and its5'-monophosphate; dNTP, deoxynucleoside triphosphate; HPLC, high-pressureliquid chromatography; THU, 3,4,5,6,-tetrahydrouridine; dTHU, 3,4,5,6-tetra-hydrodeoxyuridine; dCyd, deoxycytidine.

less, clinical cellular pharmacology studies have demonstratedthat the dFdCTP:dCTP value reaches potentially inhibitoryvalues during clinical trials (4, 10, 11). (b) dFdCTP is incorporated into DNA by DNA polymerases a and f, inhibitingfurther elongation (9). (c) Once incorporated, dFdCMP residues in the terminal or penultimate positions of the DNA strandinhibit the editing function of DNA polymerase <(9). This mayfix damage caused by the incorporated analogue, (d) dFdCDPinhibits ribonucleotide reducíase,blocking DNA synthesis bydecreasing the cellular concentrations of deoxynucleoside triphosphates (12-14).

In several human (CEM and K562) and rodent (CHO) celllines cellular elimination of high dFdCTP concentrations (>100i/\i ) is biphasic, with a short initial half-life followed by a secondphase of considerably slower degradation (7, 13, 15). Thisbiphasic elimination of dFdCTP differs from the linear kineticsexhibited during elimination of the triphosphates of arabino-sylcytosine (16), arabinosyladenine (17), and arabinosyl-2-fluo-roadenine (18) in human leukemia cells after therapy. Furthermore, the slow terminal catabolism of dFdCTP contributes toa greater dFdCTP area under the concentration x time curvein cells. The continued presence of the active nucleotides isassociated with a prolonged inhibition of DNA synthesis and,thus, may contribute to greater cytotoxicity (7, 9).

We investigated the extent to which dFdCTP elimination isaffected in CEM cells by cellular concentrations of dFdCTPand its metabolites. We suggest here a mechanistic model thatdescribes dFdC-mediated modulation of dCMP deaminase as adeterminant of dFdCTP elimination and identifies dFdC as adrug with self-potentiating activity.

MATERIALS AND METHODS

Chemicals. dFdC, dFdU, dFdCMP, and [5-14C]dFdC were synthe

sized by published procedures (1) at Lilly Research Laboratories. THUwas generously provided by Dr. Ven Narayanan, Drug Synthesis andChemistry Branch, National Cancer Institute, and dTHU was obtainedfrom Behring Diagnostics (La Jolla, CA). Deoxycytidine, dCMP,dCTP, and all other nucleosides and nucleotides were purchased fromSigma Chemical Co., Inc. (St. Louis, MO).

Synthesis of dFdCTP. dFdCTP was synthesized from dFdCMP by amodification of the procedure of Hoard and Ott (19). dFdCMP (34.3mg, 0.1 nimnl) was converted into its pyridinium salt with the pyridi-nium form of Dowex-50W X-8 cation exchange resin. The tributylam-monium salt was prepared by addition of tributylamine (2 equivalents),and the product was dried in vacuo. The residual gum was dissolved inanhydrous /V.jV-dimethylformamide (2 ml/0.1 mmol), and 1,1'-car-bonyl-bis(imidazole) (1.6 mmol) was added. After 16 h of stirring atroom temperature under argon, methanol was added (0.035 ml/0.1mmol), and the solution was stirred for IO min more. Tributylammon-ium pyrophosphate (5 equivalents, 0.5 mmol), prepared from the pyridinium salt by addition of 5 equivalents of tributylamine in N,N-dimethylformamide (5 ml/0.1 mmol), was then added dropwise. Thereaction mixture was stirred vigorously for 16 h at room temperatureand under argon. The solution was then concentrated in vacuo to 1 ml.dFdCTP was isolated by HPLC chromatography using a preparative

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aniónexchange Magnum-20 SAX column (Whatman, Inc.) using anisocratic How (10 ml/min) of 35% Buffer A (0.005 M NH4H2PO4, pH2.8) and 65% Buffer B (0.75 M NH4H2PO4, pH 3.5). The nucleotidewas adsorbed to activated charcoal, washed with H2O, and eluted withammoniacal ethanol, and the eluate was evaporated in vacuo. ThedFdCTP yield was 87%, and the purity > 95% by HPLC analysis.

Cell Line. The human T-lymphoblast cell line CCRF-CEM wasobtained from the American Type Culture Collection (Rockville, MD)and maintained in suspension culture in RPMI 1640 medium (GIBCOLaboratories, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO) at 37°Cin a humidified atmosphere

containing 5% CO2. Periodic tests for Mycoplasma contamination,conducted by the American Type Culture Collection, were consistentlynegative. All experiments were performed with exponentially growingcells. Cell number and volume were determined by a Coulter Counterequipped with a Model C-1000 particle size analyzer (Coulter Electronics, Hialeah, FL). The mean cell volume was 9.43 x 10"" liters/cell.

Nucleotide Extraction and Analysis. Cells were washed with ice-coldphosphate-buffered saline and collected by centrifugation, and the pelletwas extracted with 0.4 N HC1O4 as previously described (7, 8). Thenucleosides and nucleotides in the acid-soluble, neutralized cell extractwere analyzed by HPLC using instruments from Waters Associates,Inc. (Milford, MA). The system was equipped with two Model 6000Apumps, a Model 680 gradient programmer, and a Partisil 10-SAXaniónexchange column (250 x 4 mm) (Whatman, Inc., Clifton, NJ).The nucleotides were quantitated with a Model 440 UV detector anda Model 730 data module. A Model 490 UV detector and a Model840 data module (Waters) were used for determinations ofdeoxynucleotides.

Cellular NTP and dFdCTP were separated by HPLC using a concavegradient (Curve 9) run over 30 min at a flow rate of 3 ml/min startingwith 65% Buffer A and 35% Buffer B and ending at 100% Buffer B.External standard quantitation was used to determine the amount ofNTP detected at 280 nm of UV. The intracellular NTP concentrationwas calculated by dividing the NTP amount by the number of cellsanalyzed and the mean cell volume. Nucleoside mono-, di, -and tri-phosphates were also separated by anión exchange HPLC. A lineargradient from 100% Buffer A to 100% Buffer B was run over 40 minat a rate of 2 ml/min. To determine cellular dNTP concentrations,HClO4-soluble, neutralized extracts from 2 x IO7cells were evaporated

to dryness in an Evapomix volume-reduction apparatus (Buchler Instruments, Fort Lee, NJ), and the ribonucleotides were degraded byperiodate oxidation as previously described (20). dNTP and dFdCTPwere separated on a Partisil-10 SAX column with a total run time of43 min. An isocratic elution with 75% Buffer A and 25% Buffer C(buffer B adjusted to pH 3.7) was maintained for 20 min at a flow rateof 3 ml/min and was followed by a linear gradient to 21% Buffer A and69% Buffer C over 23 min. More than 90% of the dNTP was recoveredby this procedure, as determined by material balance measurements ofradioactive standards.

The pattern of dFdCTP elimination, correlation coefficients forgoodness of fit (r), and rrt were determined by the ESTR1P computerprogram (21).

Determination of dFdC and dFdU in the Culture Medium. The culturemedium was extracted with 0.4 N HC1O4, neutralized, and assayed byreverse-phase HPLC using a ¿iBondapakC,8 column (Waters Associates, Inc.). The nucleosides were separated by isocratic elution with0.05 M ammonium acetate, pH 6.8. The column was then regeneratedby washing with 50% methanol.

Partial Purification and Assay of dCMP Deaminase Activity. Extractsof CEM cells were prepared by a previously described method (12).dCMP deaminase was further purified by HPLC through a molecularsieve column (Protein Pak Glass 300SW; Nihon Waters, Ltd., Japan).The flow rate was 0.8 ml/min, and the buffer contained 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.5, 2 mM MgCl2,and 3 mM dithiothreitol. dCMP deaminase activity was measured bythe HPLC method reported by Fridland and Verhoef (22). The production of dUMP or dFdUMP was determined after 2, 4, and 6 min at37°C;the initial reaction rate was calculated from the slope of the

regression line. The dCMP deaminase activity was expressed as nmol

of dUMP or dFdUMP produced/mg of protein/min using bovine serumalbumin as a standard for protein measurement. The final dCMPdeaminase preparation had a specific activity of 850 nmol/mg/min.

Assay of dCMP Deaminase Activity in Intact CEM Cells. After theindicated incubation with dFdC, CEM cells (1 to 3 x IO7) wereincubated with 0.2 ^Ci of [14C]dCyd in 5 ml of cell culture medium

without fetal bovine serum for 15 min in the presence of 5 ^g ofaphidicolin (23). Cells were quickly washed with ice-cold phosphate-buffered saline and extracted with 0.4 N HC1O4. After neutralizationwith KOH, portions of the soluble extracts were analyzed using aPartisil 10-SAX column with a flow rate of 1 ml/min: 0 to 10 min,isocratic 100% Buffer A; 10 to 70 min, linear gradient from 100%Buffer A to 100% Buffer C; 70 to 75 min, isocratic 100% Buffer C.Radioactive deoxynucleotides were detected with a radioactive flowdetector (Model A250; Packard Instrument Co., Meriden, CT). Theeluant was mixed with scintillation fluid (Flo-Scint, IV; Packard Instrument Co) at a 1:3 ratio. The dCMP deaminase activity index wascalculated using the following equation

[14C]dTTP (dpm)|['4C]dCTP (dpm) + [HC]dTTP (dpm)|

Measurement of Cellular dCTP and dTTP Pools. Deoxyribonucleo-side triphosphates were extracted from CEM cells with 0.4 N HC1O4,and the acid-insoluble material was removed by centrifugation. Thesupernatant was carefully monitored to pH 7 with KOH, and afterremoval of KC1O4, samples were stored at -20°C until analysis. A

DNA polymerase assay using synthetic oligonucleotides as templateprimers was applied to determine dCTP and dTTP pools as describedby Sherman and Fyfe (24).

RESULTS

dFdCTP Accumulation. dFdCTP accumulated rapidly inCEM cells incubated with 0.01 to 10 ^M dFdC. As shown inFig. 1, increasing the concentration of exogenous nucleosideproportionally increased the triphosphate. Cellular dFdCTPconcentrations as great as 500 ^M accumulated after cells wereincubated with 10 MMdFdC for 2 h.

Cellular dFdCTP Elimination. The relationship between cellular dFdCTP concentration and the rate of dFdCTP elimination was analyzed after a 2-h incubation of CEM cells with 0.1,0.3, 1.0, and 10 MMdFdC (Fig. 2). Average cellular dFdCTPconcentrations of 37, 138, 346, and 525 MM accumulated,respectively. After the cells were washed into drug-free culturemedium, the patterns of dFdCTP elimination were determined.Cellular elimination of dFdCTP was linear with a />/,of 3.0 hafter a 2-h incubation with 0.1 MMdFdC. In contrast, after

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Fig. 1. dFdCTP accumulation in CEM cells. Cells were incubated with theindicated concentrations of dFdC for 2 h, when nucleotide pools were extractedand analyzed for dFdCTP content. Points, mean; bars, SEM.

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Fig. 2. dFdCTP elimination as a function of cellular dFdCTP concentration.CEM cells were incubated for 2 h with 0.1 (•),0.3 (A), 1.0 (»),or 10 (O) MMdl (K - After washing cells into drug-free medium, cellular concentrations ofdFdCTP were measured at the indicated times. Correlation coefficients (r) forTilting of elimination kinetics were the following: 0.1 MM,0.964; 0.3 /.\i. 0.99S;1.0 MM,0.946; 10 MM,0.970. Points, mean of three separate experiments. Standarddeviations, which were omitted for clarity, were less than 15% of the means.

incubation with 0.3,1.0, and 10 MMdFdC, dFdCTP eliminationwas best fit to biphasic kinetics; the initial tv, values of 0.6, 1.2,and 1.3 h, respectively, were not significantly different. Theterminal tv, values of dFdCTP in these cultures, 5, 16, and 43h, respectively, increased as a function of cellular dFdCTPconcentration. Cellular integrity, as determined by Coulter volume analysis, was maintained throughout the experiments.

dFdC Metabolites. The intracellular distribution of dFdCmetabolites in CEM cells was determined after a 2-h incubationwith either 0.1 MMor 10 MMdFdC (Table 1) to investigate themetabolic basis for the concentration-dependent elimination ofdFdCTP. Similar percentages of dFdC metabolites were observed with the two concentrations. dFdCTP was the majordrug metabolite (about 80% of the total) followed by dFdUMP(about 15%), dFdCDP (about 2%), and dFdCMP (about 1%).The percentage of dFdCTP decreased with increasing time afterwashout as did the ratio of dFdU to dFdUMP. Although dFdUrose approximately 10-fold in cells from each culture, it comprised less than 10% of the cellular dFdC metabolites at 8 h.However, dFdUMP levels nearly doubled to >20% of cellulardFdC metabolites as the percentage of dFdCTP decreased inthe cells with low initial dFdCTP concentrations.

Excretion of dFdC and dFdU. To better characterize the endproducts of dFdCTP catabolism, we analyzed the culture medium for dFdC and its metabolites after a 2-h incubation with0.1 MMor 10 MM[14C]dFdC. dFdC and dFdU were the onlyradioactive compounds detected after washing cells into drug-free medium. After incubation with 0.1 MMdFdC, dFdU accumulated in the medium to a substantially greater extent thandid dFdC (Fig. 3A). The accumulation of dFdU was linear overthe initial 4 h (1.8 pmol/ml x h) and then reached a plateau,whereas the dFdC level detected in the medium remained atessentially background levels after drug washout. In contrast,accumulation of dFdC in the medium greatly exceeded that ofdFdU after incubation with 10 MMdFdC (Fig. 3B). At 4 h afterdrug washout, when dFdC in the medium reached a maximum,the ratio of dFdU to dFdC was 21% to 79%. At that time dFdCaccumulation reached a plateau, reflecting the low rate ofdFdCTP elimination (Fig. 3). dFdU accumulation (3.1 pmol/ml x h) remained linear over 8 h after drug washout. Thus, athigh cellular dFdCTP concentrations, dephosphorylation andexcretion of dFdC appeared to be the dominant catabolic route,

whereas at low cellular dFdCTP levels, deaminationpredominated.

The activity of deoxycytidine deaminase is very low in CEMcells (22); this suggests that the dCMP deaminase pathway ismainly responsible for dFdCMP deamination. Additional evidence was sought to reveal the role this enzyme plays indFdCTP elimination. CEM cells were incubated for 2 h with0.1 MM [l4C]dFdC, followed by washing into fresh medium

alone, with 100 MMdTHU or 100 MMTHU. After 4 h, theproportions of dFdC and dFdU in the medium containingcontrol cells were 32% to 68%, respectively. This did not changein the culture to which the cytidine deaminase inhibitor, THU,was added (33 to 67%, respectively). Excretion of dFdU waslargely inhibited, however, in cells incubated with dTHU (88%dFdC to 12% dFdU, respectively). It is worth noting that dTHUinhibits cytidine deaminase and, after intracellular phosphor-ylation to the monophosphate, it is a potent inhibitor of dCMPdeaminase (25). These results are consistent with the notionthat dCMP deaminase has a central role in dFdCTP elimination. Based on the foregoing data, we tested the hypothesis thatdeamination was the rate-limiting step in dFdCTP elimination

and that high concentrations of dFdC metabolites may either

Table 1 Cellular distribution ofdFdC metabolitesCEM cells were incubated for 2 h with either 10 MM[MC]dFdC or 0.1 MM[MC]

dFdC. The cellular dFdCTP levels were 464 MMand 38 MM,respectively. Cellswere then washed into fresh medium and portions of each culture were extractedat the indicated times and analyzed for intracellular dFdC metabolites.

% of total metabolites

Oh 2b 41, 6h 8h10 MMdFdCmetabolitedFdCdFdUdFdCMPdFdUMPdFdCDPdFdCTP0.1

MMdFdCmetabolitedFdCdFdUdFdCMPdFdUMPdFdCDPdFdCTP0.30.41.415.31.880.9ND"0.90.411.92.384.50.60.83.115.76.173.6ND1.41.413.92.980.50.61.44.217.74.971.2ND3.22.417.53.473.51.24.41.915.23.074.3ND4.93.417.42.471.83.62.216.03.076.3ND8.32.420.51.667.2

1ND, not detected.

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Fig. 3. Extracellular accumulation of dFdU and dFdC as catabolites of dFdCTPelimination. CEM cells were exposed to 0.1 MM(14C|dFdC (A) or to 10 MM[UC]dFdC (B) for 2 h and washed into drug-free medium. At the indicated times,dFdU (O) and dFdC (•)concentrations in the medium were determined. Points.mean of two experiments; bars, SEM.

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MM,dFdC, CEM cells were washed free from the drug and were reincubated with100 ¡IMdTHU (O) or without dTHU (•).The cellular dFdCTP concentrationswere determined at the indicated time points. Points, mean of three separateexperiments; bars, SEM. Occasionally a SEM was less than the size of the symbol.

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Fig. S. Inhibition of dCMP deaminase by dFdC after drug washout. CEM cellswere preincubated with 0 (O), 0.1 (•),0.3 (A), 1.0 (X), or 10.0 (D) MMdFdC for2 h. Cells were then washed into drug-free medium, and the dCMP deaminaseactivity index (A) and dCTP:dTTP values (B) were measured. Points mean of twoseparate experiments.

directly or indirectly inhibit dCMP deaminase, decreasing excretion of dFdU into the medium.

dCMP Deaminase as a Determinant of dFdCTP Elimination.To evaluate the mechanism of the concentration-dependentelimination characteristics of dFdCTP, we used dTHU to inhibit dCMP deaminase activity in intact cells. After incubationwith 0. l MMdFdC for 2 h, the cells were washed into drug-freemedium. As shown previously, the cellular dFdCTP concentration reached about SO ¿<Mand, after washing into drug-freemedium, dFdCTP elimination was linear (f,/,= 3.6 h, r = 0.990)(Fig. 4). A parallel culture was treated identically except that100 MMdTHU was added after the cells were resuspended infresh medium. dFdCTP elimination in these cells was biphasic,with an apparent terminal i./,of 19 h (r = 0.950). The inflectionfrom linear elimination kinetics occurred 4 to 5 h after drugwashout. This effect could not be achieved in a third culture byaddition of THU; no deviation from linear dFdCTP eliminationwas apparent (/./, = 4.5 h; data not shown).

Inhibition of dCMP Deaminase In Situ. The effect of dFdCon dCMP deaminase activity was assayed in whole cells byfollowing the incorporation of radioactivity from added [14C]

dCyd into dTTP. CEM cells were incubated for 2 h with either

0.1 to 10 MMdFdC before being washed into drug-free medium.dCMP deaminase activity in situ was measured in indicatedtime periods after drug washout (Fig. 5A). dCMP deaminasewas inhibited in a dose-dependent manner. After washing intodrug-free medium, the enzyme activity remained suppressed incells treated with 1 and 10 MMdFdC, but recovered in cellstreated with 0.3 and 0.1 MMdFdC. The dCTP and dTTP poolswere also measured in these cells. The dCTP:dTTP value incontrol cells was 0.38, whereas this decreased to 0.15, 0.11,0.08, and 0.04 after the 2-h incubation with 0.1, 0.3, 1.0, and10 MMdFdC, respectively (Fig. 5B). As with inhibited dCMPdeaminase activity, recovery of the cellular dCTP:dTTP valuesafter dFdC washout was dose dependent.

Substrate Specificity of dCMP Deaminase. The ability ofdCMP deaminase to deaminate dFdCMP, dCMP, and 1-/3-D-arabinofuranosylcytosine monophosphate was assayed in partially purified extracts of CEM cells. The apparent Km valueswere 0.138 ±0.003, 0.061 ±0.003, and 1.062 ±0.483 mM,respectively. The apparent Fmaxvalues were 6.1 ±0.17, 27.1 ±2.4, and 2.0 ±0.6 Mmol/mg/min, respectively, yielding respective substrate efficiencies ( Kmax:/Tm)of 44,444, and 2. Thus, theeffectiveness of dCMP deaminase-mediated deamination ofdFdCMP appears to be 10-fold less than that of dCMP but 22-fold greater than l-/3-D-arabinofuranosylcytosine monophos-phate deamination.

Modulation^ dFdCMP Deamination by dCTP and dTTP. Itis well established that the activity of dCMP deaminase fromother sources (26, 27) on dCMP is regulated by dCTP anddTTP. A similar relationship appeared to hold for the deamination of dFdCMP. In the absence of dCTP no dFdCMPdeamination was evident (Fig. fi.-l). Although their absoluteactivities differed, dCTP-stimulated deamination of dCMP anddFdCMP exhibited similar patterns of activation. dTTP inhibited dCMP deaminase activity on both dCMP and dFdCMP(Fig. 6B). The dTTP:dCTP value was critical to dCMP deaminase activity on dFdCMP. At 25 MM dCTP, 500 MM wasnecessary to inhibit dFdCMP deamination 90%. In contrast, at5 MMdCTP, dFdCMP deamination was inhibited >90% by 100MM dTTP. dFdCMP deamination was inhibited 50% whendTTP:dCTP = 4. Maximum inhibition of dCMP deaminasewas reached in both cases at a dTTP:dCTP value of 20, regardless of the absolute concentrations.

dFdCTP Inhibition of dCMP Deaminase. Because dFdC536

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METABOLIC SELF-POTENTIATION OF dFdC

deamination was dependent on the dFdCTP concentration, wesought to determine whether dFdCTP might directly inhibitdCMP deaminase activity. The preparation of dCMP deami-nase from CEM cells was inhibited by dFdCTP (Fig. 7). In thepresence of 5 ^M dCTP, deamination of dCMP was inhibitedwith 50% inhibitory concentration of 0.46 mM dFdCTP. Asshown in Fig. 1, this cellular concentration is exceeded afterincubation with 10 UMdFdC for 2 h.

DISCUSSION

Cellular dFdCTP elimination is a concentration-dependentprocess with biphasic kinetics at cellular levels of 100 pMdFdCTP or greater (Fig. 2). Lower dFdCTP concentrations (50MM)were eliminated with linear kinetics after cells were washedinto fresh medium. Although the initial tVlwas apparently notinfluenced by cellular concentrations of dFdCTP, the terminalivi increased with increasing cellular dFdCTP concentration;elimination was almost completely inhibited at 300 /¿MdFdCTP. Comparable dFdCTP elimination kinetics valueswere found in K562 (13), Chinese hamster ovary (7), and HL-60 cells (data not shown). The concentration-dependent prolongation of the terminal !•is associated with an increased areaunder the concentration x time curve of cellular dFdCTP andthus may enhance cytotoxicity (7, 9).

Accumulation of intermediary metabolites might indicatewhich specific step was responsible for the differential elimination kinetics of dFdCTP. The cellular distribution of accumulated dFdC metabolites was not much different among cellcultures incubated 2 h with 0.1 and 10 UM dFdC (Table 1).However, 8 h after washing into fresh medium, the amount ofdFdUMP doubled in cells containing low concentrations ofdFdCTP, whereas dFdUMP in cells incubated with 10 ^M dFdCdid not increase substantially. dFdUMP was a major cellularproduct of dFdCTP catabolism under either condition. BecausedFdU is a poor substrate for phosphorylation to dFdUMP (13),it may be assumed that dFdUMP arose from the action ofdCMP deaminase.

To pursue this possibility, the extracellular medium wasanalyzed for the catabolic end products dFdC and dFdU. Excretion of nucleosides into the culture medium after drug washout initially occurs in a primarily unidirectional fashion. Thus,reequilibration of intracellular and extracellular nucleosides isinitially not likely to confound the analysis. Although dFdC isa good substrate for dCyd deaminase (13), the activity of thisenzyme in CEM cells is extremely low (22). Extracellularaccumulation of dFdU is therefore a result of dCMP deaminaseactivity and can be suppressed by dTHU. Because dFdU wasnot significantly phosphorylated in CEM cells, extracellulardFdU may be regarded as an end product of dFdCTP catabolism arising from the activity of dCMP deaminase.

At low intracellular dFdCTP concentrations (Fig. 3/1), dFdUwas the predominant metabolite in the extracellular medium.However, at high cellular dFdCTP concentrations (Fig. 3Ä),relatively low levels of dFdU were excreted; the major extracellular metabolite was dFdC. It appears that, at high dFdCTPconcentrations, the activity of dCMP deaminase was significantly less important than dephosphorylation of dFdCMP todFdC. Thus, dFdC accumulated extracellularly as the predominant product. These findings may be due to diminished dCMPdeaminase activity at high cellular concentrations of dFdCTP.

Two mechanisms of dFdC-mediated inhibition of dCMPdeaminase were observed, (a) dCMP deaminase is subject to

100

80

40

20

0.0 0.5 1.0

dFdCTP. mM

Fig. 7. Inhibition of dCMP deamination by dFdCTP. dCMP deamination wasmeasured in partially purified cell extracts by determining the amount of dUMPformed. The experiment was performed with 61 /IM dCMP as a substrate fordCMP deaminase in the presence of 5 JIMdCTP. Points, mean of three determinations; bars, SEM.

allosteric regulation by dCTP and dTTP (26, 27). dCTP actsas an allosteric activator of dCMP deaminase-mediateddFdCMP deamination, whereas dTTP inhibits this reaction(Fig. 6); the degree of inhibition was dependent on thedCTP:dTMP value. The importance of these considerations isevident in view of dFdC-mediated perturbations of cellulardNTPs (Fig. 5B). dFdCDP is a potent inhibitor of ribonucleo-tide reducíase(12-14). dFdC decreased dCTP to a significantlygreater extent than dTTP, thus lowering the value ofdCTP:dTTP which probably contributed to dFdC-mediatedinhibition of dCMP deaminase. The degree of dCTP:dTTPdecrease and the rate of recovery from the decrease were correlated with the cellular concentration of dFdCTP and itsmetabolites, (b) dFdCTP may consequently inhibit its owncatabolism by a concentration-dependent modulation of dCMPdeaminase. dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. Intracellular dFdCTP concentrations close to this 50% inhibitory concentration value were reached after a 2-h incubation with 10MMdFdC (Fig. 2). It may therefore be assumed that dFdCMPdeamination is partly inhibited in the cell by high dFdCTPconcentrations.

We tested the hypothesis that dFdC mediates dCMP deaminase inhibition with regard to the kinetics of dFdCTP elimination using THU as a selective inhibitor of dCyd deaminase.dTHU, which also inhibits dCyd deaminase, is phosphorylatedto dTHUMP, which is an inhibitor of dCMP deaminase (25).dTHU-mediated inhibition of dCMP deaminase and dCyd deaminase changed linear dFdCTP elimination to biphasic elimination kinetics similar to that seen at high dFdCTP concentrations (Fig. 4). This result could not be obtained by THU, whichsolely inhibits dCyd deaminase. Accordingly, we hypothesizedthat deflection from linear dFdCTP elimination was primarilydue to inhibition of dCMP deaminase. Inhibition of dFdCMPdeamination would decrease dFdCTP catabolism, contributingto the change in the elimination kinetics of dFdCTP.

Direct measurement of dCMP deaminase activity in wholecells confirmed that the enzyme was inhibited by preincubationwith dFdC (Fig. 6A). The inhibition was dependent on exogenous dFdC concentration. Higher intracellular dFdCTP concentrations apparently resulted in stronger inhibition of dCMPdeaminase activity. This was most likely the combined effect ofdirect inhibition of the enzyme by dFdCTP and an indirect

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METABOLIC SELF-POTENTIATION OF dFdC

dFdC dFdU

Fig. 8. Scheme of cellular metabolism and actions of dFdC and its nucleotides.Dashed lines indicate inhibitory actions.

action brought about by the decrease of the dCTP:dTTP value.Like most of the dFdC metabolites, dCMP deaminase activityremained relatively unchanged up to 8 h after washing the cellstreated with 10 UMdFdC into fresh medium.

A summary of the cellular metabolism of dFdC and theactions of its nucleotides is presented in Fig. 8. The presentstudies will be discussed with the view that the pharmacody-namic actions of dFdC nucleotides potentiate the cytotoxicityof the drug, (a) The major cellular metabolite, dFdCTP, competes with dCTP for incorporation into DNA by DNA poly-merases (9) (Fig. 8, Reaction 1). Although dFdCMP is not anabsolute DNA chain terminator for DNA polymerase a or e,its incorporation causes a pause in the replication rates of theseenzymes. Once incorporated, it is not removed by the 3' —¿�»5'

proof-reading exonuclease of DNA polymerase e, and its incorporation is strongly correlated with loss of clonogenicity (9).(b) dNTP pools decrease in cells treated with dFdC (12, 13),an action that appears to be due to inhibition of ribonucleosidediphosphate reducíaseby dFdCDP (12, 14) (Fig. 8, Reaction2). This action decreases the cellular concentration of dCTPand thus, its ratio with dFdCTP. As demonstrated in purifiedenzyme systems (9), this is likely to increase the incorporationof dFdCTP into DNA and to potentiate its cytotoxicity. (c)Deoxycytidine kinase is required for dFdC phosphorylation (7,8) and is rate limiting to the accumulation of dFdCTP (8, 10,11) (Fig. 8, Reaction 3). Because the activity of deoxycytidinekinase is regulated by dCTP (28, 29), it is likely that thedFdCDP-induced decrease in dNTP pools results in activationof the enzyme and a more rapid phosphorylation of dFdC. Theresulting increased dFdCTP accumulation and incorporationinto DNA would be viewed as another action that potentiatesthe cytotoxicity of dFdC. (d) Deamination of dFdCMP bydCMP deaminase requires activation by dCTP (Fig. 8, Reaction4); the lowering of the dCTP pool is associated with a decreasein the enzyme activity. Because this is a major pathway of dFdCnucleotide elimination, inhibition of dCMP deaminase by theaction of dFdCDP on ribonucleotide diphosphate reducíasewould prolong the retention of dFdC nucleotides, an actionassociated with increased cytotoxicity. (e) Finally, at relativelyhigh concentrations, dFdCTP is a direct inhibitor of dFdCMPdeamination by dCMP deaminase (Fig. 8, Reaction 5). Combined with the dFdCDP-induced reduction in the dCTP pools,this action may cause the concentration-dependent eliminationkinetics of dFdCTP in CEM cells. The relatively prolongedretention of dFdC nucleotides, which has recently been observedin circulating leukemia cells during dFdC therapy (30), may bethe pharmacological basis for the activity of this drug in experimental systems (2, 3) and in clinical trials (4-6).

ACKNOWLEDGMENTS

The authors are grateful to Maureen Goode for valuable editorialassistance in the preparation of this manuscript.

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1992;52:533-539. Cancer Res   Volker Heinemann, Yi-Zheng Xu, Sherri Chubb, et al.   -Triphosphate: A Mechanism of Self-Potentiation

′-Difluorodeoxycytidine 5′,2′Cellular Elimination of 2

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