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Difluorodeoxycytidine 5'-Triphosphate: a Mechanism of Self-Potentiation1

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Difluorodeoxycytidine 5'-Triphosphate: a Mechanism of Self-Potentiation1 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 Research Laboratories, Indianapolis, Indiana 46285 (L. W. H., G. B. G.] ABSTRACT less, clinical cellular pharmacology studies have demonstrated 2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine that the dFdCTP:dCTP value reaches potentially inhibitory analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate values during clinical trials (4, 10, 11). (b) dFdCTP is incor porated into DNA by DNA polymerases a and f, inhibiting (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and further elongation (9). (c) Once incorporated, dFdCMP resi found they were dependent on cellular concentration after incubation of dues in the terminal or penultimate positions of the DNA strand CCRF-CEM cells with dFdC and washing into drug-free medium. When inhibit the editing function of DNA polymerase <(9). This may the drug was washed out at low cellular dFdCTP levels (<50 n\\), fix damage caused by the incorporated analogue, (d) dFdCDP dFdCTP elimination was linear (t,: = 3.3 h), but it became biphasic at inhibits ribonucleotide reducíase,blocking DNA synthesis by intracellular dFdCTP levels >100 JIM. Although the initial elimination decreasing the cellular concentrations of deoxynucleoside tri rate was similar at all concentrations, at higher concentrations the phosphates (12-14). terminal elimination rate increased with increasing cellular dFdCTP In several human (CEM and K562) and rodent (CHO) cell concentration, with a nearly complete inhibition of dFdCTP elimination at 300 pM. The deamination product 2',2'-difluorodeoxyuridine was the lines cellular elimination of high dFdCTP concentrations (>100 i/\i ) is biphasic, with a short initial half-life followed by a second predominant extracellular catabolite at low cellular dFdCTP concentra tions, whereas at high dFdCTP concentrations dFdC was the major phase of considerably slower degradation (7, 13, 15). This excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydro- biphasic elimination of dFdCTP differs from the linear kinetics deoxyuridine transformed the monophasic dFdCTP degradation seen at exhibited during elimination of the triphosphates of arabino- low dFdCTP levels into a biphasic process, whereas the deoxycytidine sylcytosine (16), arabinosyladenine (17), and arabinosyl-2-fluo- deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP roadenine (18) in human leukemia cells after therapy. Further elimination. An in situ assay indicated that dCMP deaminase activity more, the slow terminal catabolism of dFdCTP contributes to was inhibited in whole cells, an action that was associated with a a greater dFdCTP area under the concentration x time curve decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially in cells. The continued presence of the active nucleotides is purified dCMP deaminase with a 50% inhibitory concentration of 0.46 m\i. We conclude that dFdC-induced inhibition of dCMP deaminase associated with a prolonged inhibition of DNA synthesis and, thus, may contribute to greater cytotoxicity (7, 9). resulted in a decrease of dFdCTP catabolism, contributing to the concen tration-dependent elimination kinetics. This action constitutes a self- We investigated the extent to which dFdCTP elimination is potentiation of dFdC activity. affected in CEM cells by cellular concentrations of dFdCTP and its metabolites. We suggest here a mechanistic model that describes dFdC-mediated modulation of dCMP deaminase as a INTRODUCTION determinant of dFdCTP elimination and identifies dFdC as a dFdC4 (Gemcitabine) is a deoxycytidine analogue in which drug with self-potentiating activity. geminai fluorines replace both hydrogens of the 2' carbon atom (1). An unusually broad spectrum of activity in murine tumors MATERIALS AND METHODS (2) and human tumor xenografts (3) provided impetus for Chemicals. dFdC, dFdU, dFdCMP, and [5-14C]dFdC were synthe evaluation of its anticancer activity in clinical trials (4-6). dFdC sized by published procedures (1) at Lilly Research Laboratories. THU must be phosphorylated by deoxycytidine kinase to exhibit was generously provided by Dr. Ven Narayanan, Drug Synthesis and cytotoxic and therapeutic activities (7,8). Its major intracellular Chemistry Branch, National Cancer Institute, and dTHU was obtained metabolite is dFdCTP, although it remains in a constant ratio from Behring Diagnostics (La Jolla, CA). Deoxycytidine, dCMP, with the lesser metabolites, dFdCMP and dFdCDP (7). dCTP, and all other nucleosides and nucleotides were purchased from DNA synthesis is specifically inhibited by dFdC by several Sigma Chemical Co., Inc. (St. Louis, MO). separate mechanisms, (a) dFdCTP competes with dCTP as a Synthesis of dFdCTP. dFdCTP was synthesized from dFdCMP by a weak inhibitor of mammalian DNA polymerase (9). Neverthe- modification of the procedure of Hoard and Ott (19). dFdCMP (34.3 mg, 0.1 nimnl) was converted into its pyridinium salt with the pyridi- Received 8/21/91; accepted 11/13/91. nium form of Dowex-50W X-8 cation exchange resin. The tributylam- The costs of publication of this article were defrayed in part by the payment monium salt was prepared by addition of tributylamine (2 equivalents), of page charges. This article must therefore be hereby marked advertisement in and the product was dried in vacuo. The residual gum was dissolved in accordance 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 anhydrous /V.jV-dimethylformamide (2 ml/0.1 mmol), and 1,1'-car- Grant CA28596 from the National Cancer Institute, Department of Health and bonyl-bis(imidazole) (1.6 mmol) was added. After 16 h of stirring at Human Services. : Present address: Department of Internal Medicine. Hematology/Oncology, room temperature under argon, methanol was added (0.035 ml/0.1 mmol), and the solution was stirred for IO min more. Tributylammon- Klinikum Gro¡ihadcrn,University of Munich, Munich. Germany. 3To whom requests for reprints should be addressed. ium pyrophosphate (5 equivalents, 0.5 mmol), prepared from the pyri 4 The abbreviations used are: dFdC, 2'.2'-difluorodeoxycytidine;CEM, CCRF- dinium salt by addition of 5 equivalents of tributylamine in N,N- CEM lymphoid cells; dFdCMP, dFdCDP, and dFdCTP, the 5'-mono-, dì-,and triphosphates of dFdC; dFdU and dFdUMP, 2'.2'-difluorodeoxyuridinc and its dimethylformamide (5 ml/0.1 mmol), was then added dropwise. The 5'-monophosphate; dNTP, deoxynucleoside triphosphate; HPLC, high-pressure reaction mixture was stirred vigorously for 16 h at room temperature liquid chromatography; THU, 3,4,5,6,-tetrahydrouridine; dTHU, 3,4,5,6-tetra- and under argon. The solution was then concentrated in vacuo to 1 ml. hydrodeoxyuridine; dCyd, deoxycytidine. dFdCTP was isolated by HPLC chromatography using a preparative 533 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. METABOLIC SELF-POTENTIATION OF dFdC aniónexchange Magnum-20 SAX column (Whatman, Inc.) using an of dUMP or dFdUMP produced/mg of protein/min using bovine serum isocratic How (10 ml/min) of 35% Buffer A (0.005 M NH4H2PO4, pH albumin as a standard for protein measurement. The final dCMP 2.8) and 65% Buffer B (0.75 M NH4H2PO4, pH 3.5). The nucleotide deaminase preparation had a specific activity of 850 nmol/mg/min. was adsorbed to activated charcoal, washed with H2O, and eluted with Assay of dCMP Deaminase Activity in Intact CEM Cells. After the ammoniacal ethanol, and the eluate was evaporated in vacuo. The indicated incubation with dFdC, CEM cells (1 to 3 x IO7) were dFdCTP yield was 87%, and the purity > 95% by HPLC analysis. incubated with 0.2 ^Ci of [14C]dCyd in 5 ml of cell culture medium Cell Line. The human T-lymphoblast cell line CCRF-CEM was without fetal bovine serum for 15 min in the presence of 5 ^g of obtained from the American Type Culture Collection (Rockville, MD) aphidicolin (23). Cells were quickly washed with ice-cold phosphate- and maintained in suspension culture in RPMI 1640 medium (GIBCO buffered saline and extracted with 0.4 N HC1O4. After neutralization Laboratories, Grand Island, NY) supplemented with 10% heat-inacti with KOH, portions of the soluble extracts were analyzed using a vated fetal bovine serum (GIBCO) at 37°Cina humidified atmosphere Partisil 10-SAX column with a flow rate of 1 ml/min: 0 to 10 min, containing 5% CO2. Periodic tests for Mycoplasma contamination, isocratic 100% Buffer A; 10 to 70 min, linear gradient from 100% conducted by the American Type Culture Collection, were consistently Buffer A to 100% Buffer C; 70 to 75 min, isocratic 100% Buffer C. negative. All experiments were performed with exponentially growing Radioactive deoxynucleotides were detected with a radioactive flow cells. Cell number and volume were determined by a Coulter Counter detector (Model A250; Packard Instrument Co., Meriden, CT). The equipped with a Model C-1000 particle size analyzer (Coulter Electron eluant was mixed with scintillation fluid (Flo-Scint, IV; Packard In ics, Hialeah, FL). The mean cell volume was 9.43 x 10"" liters/cell. strument Co) at a 1:3 ratio. The dCMP deaminase activity index was Nucleotide Extraction and Analysis. Cells were washed with ice-cold calculated using the following equation phosphate-buffered saline and collected by centrifugation, and the pellet [14C]dTTP (dpm) was extracted with 0.4 N HC1O4 as previously described (7, 8). The |['4C]dCTP (dpm) + [HC]dTTP (dpm)| nucleosides and nucleotides in the acid-soluble, neutralized cell extract were analyzed by HPLC using instruments from Waters Associates, Measurement of Cellular dCTP and dTTP Pools.
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