[CANCER RESEARCH55, 1517-1524, April 1, 19951 Cytotoxicity, Metabolism, and Mechanisms of Action of 2',2'- Difluorodeoxyguanosine in Chinese Hamster Ovary Cells' Varsha Gandhi,2 Shin Mineishi, Peng Huang, Amy J. Chapman, Yandan Yang, Feng Chen, Biffie Nowak, Shern Chubb, Larry W. Hertel, and William Plunkett Department of Clinical Investigation, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 (V. G., S. M., P. H.. A. J. C.. Y. 1'., F. C., B. N., S. C., w. p.]. andLillyResearchLaboratories,Indianapolis,indiana46285(L W.H.) ABSTRACT treatment of hematological malignancies (4—6).Arabinosyladenine, although not successful in cancer therapy, has proven antiviral activity The emerging clinical success of gemcItabine (2',2'-difluorodeoxycytl dine) Stimulated interest in the synthesis and evaluation of purine conge (7). The importance of the 2'-pentose moiety for the design of chemo ners. The cytotoxicity, metabolism, and mechanisms of action of the lead therapeutic agents and the fact that fluorine has a van der Waals radius candidate, 2',2'-difluorodeoxyguanoslne (dFdGuo), were StUdied In ChI nese hamster ovary cells Unlike the natural nucleoside deoxyguanosine similar to that of hydrogen led to the synthesis and evaluation of (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase 2'-fluorinated analogues. 2'-Deoxy-2'-fluorocytidine and 2'-deoxy Wild-type Chinese hamster ovary cells and a mutant line deficient in 2'-fluoroguanosine possess inhibitory activities against several deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% lymphoid cell lines and influenza viruses, respectively (8, 9). Inter inhibitory concentration, 7.5 and 6.5 pM, respectively), suggesting that estingly, substitution of the 2'-hydrogen of dCyd3 with a fluorine unlike gemcitabine, dCyd kinase was not responsible for activation of atom in the arabinose configuration produced 10-fold greater cyto dFdGuo This was further confirmed by separation of nucleoside kinases toxicity than when placed in the ribose position (10). 2'-Fluoroarabi (adenosine kinase, dGuo ldnase, and dCyd kinase) of Chinese hamster nosylguanine is active against human leukemia cells in vitro (11). ovary cells on DEAE-cellulose column chromatography. The kinase ac Recently, substitution of both hydrogens of the 2'-carbon of dCyd tivity that phosphorylated dGuo also converted dFdGuo to its monophos. with fluorines generated dFdCyd (12), which is currently being phate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; evaluated in the clinic under the generic name gemcitabine. however, addition of up to 10 mM dCyd did not reverse the toxicity of Comparative studies between ara-C and dFdCyd demonstrated that dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, the difluoro anomer is transported more rapidly into cells and is a and triphosphates; dFdGuo triphosphate (dFdGTP) was the major me preferred substrate for phosphorylation to the active 5'-triphosphate, tabolite and accumulated to 45 jiM after a 6-h Incubation with 30 jaM which is relatively stable in cells (i3). Additionally, unlike ara-C, the dFdGuo. The elimination of dFdGTP was monophasic with a t½ofabout diphosphate of dFdCyd inhibited ribonucleotide reductase, an action 6 h. Deoxynucleotides were decreased in cells Incubated with dFdGuo, that decreased deoxynucleotide pools and was the key to the suggesting that ribonudeotide reductase was Inhibited. dAT?, which de self-potentiation of the analogue (14—16). creased 78% after a 4-h Incubation with 30 jaM dFdGuo, was most This multitude of actions of gemcitabine metabolites in exerting affected@dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA printer over a defined template in the presence of dFdGTP revealed cytotoxicity and its emerging clinical success (17) stimulated interest that dFdGTP was a good substrate for incorporation opposite C sites of in the synthesis and evaluation of purine congeners (18, 19). Meta the template by DNA polymerase a dFdGTP incorporation caused DNA bolic inactivation and growth inhibition studies in a variety of tumor polymerase a to pause after the polymerization of one additional de lines suggested that dFdGuo (Fig. 1) was the lead analogue among the oxynucleotide. This pattern oflnhlbltlon, which is shared by gemcltabine, purine series (18, 19). Using CHO cells, we investigated the cytotox distinguishes 2',2'-difluoronucleosldes from arabinosylnudeosides which icity, metabolism, and mechanisms of action of dFdGuo. Emphasis is halt primer extension at the Incorporation slte dGTP competed effectively placed on the enzymes likely to be involved in the catabolism (purine with dFdGTP for incorporation by DNA polymerase a The unique nucleoside phosphorylase) and anabolism (nucleoside kinases) of activation requirements and patterns of Inhibition of DNA synthesis din dFdGuo and on ribonucleotide reductase and DNA polymerase, which tinguish this promising new antimetaboilte from other nucleoside by analogy with dFdCyd are likely targets of dFdGuo nucleotides. The analogues. results are compared with those of dFdCyd and the base congener ara-G. INTRODUCTION MATERIALS AND METHODS The chemical constituents and their configuration at the 2'-position of purine and pynmidine nucleotides are critical determinants of the Chemicals. dFdGuo, its 5'-triphosphate, dFdGTP, and dFdlno were syn metabolic fate and therapeutic activity of these nucleic acid precur thesized at the Lilly Research Laboratories, Indianapolis, IN. [8-3HJdFdGuo sors. A hydroxyl group in the arabinose configuration results in the (specific activity, 6 Ci/mmol) was prepared by Amersham Radiochemicals, incorporation of these arabinosyl nucleosides into DNA and inhibition Arlington, IL. Bovine spleen purine nucleoside phosphorylase was purchased of further DNA synthesis, leading to cell death (1—3).Arabinosyl from Sigma Chemical Co., St. Louis, MO, as were natural nucleosides and analogues of both purine and pyrimidine are established agents in the nucleotides. All other chemicals were of the highest purity available. For nucleoside kinase assays, the natural nucleosides [2,8-3H]adenosine(39 Ci/mmol), [5-3H]dCyd (26 Ci/mmol), and [8-3H]dGuo (7 Ci/mmol) were Received 11/29/94; accepted 2/3/95. Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage charges. This article must therefore be hereby marked advertisement in accordance with 3 The abbreviations used are: dCyd, deoxycitidine; dAdo, deoxyadenosine; ara-C and 18 U.S.C. Section 1734 solely to indicate this fact. ara-cm', 1-@-t-arabinofuranosylcytosineandits 5'-triphosphate;ara-Gand ara-GTP, 1 Supported in part by Grants CA 28596 and CA 57629 from the National Cancer i-@-D-arabinofuranosylguanine and its 5'-triphosphate; dFdCyd, dFdCMP, dFdCDP, and Institute, Department of Health and Human Services, and Grant DHP-1 from the Amer dFdCFP, 2',2'-difluorodeoxycytidine (gemcitabine) and its 5'-mono-, -di-, and -triphos icanCancerSociety. phate;dFdGuo,dFdGMP,dFdGDP,anddFdGTP,2',2'-difluorodeoxyguanosineandits 2 To whom requests for reprints should be addressed, at Department of Clinical 5'-mono-, -di-, and -triphosphate; dGuo, deoxyguanosine; dFdlno, difluorodeoxyinosine; Investigation, Box 52, The University of Texas M. D. Anderson Cancer Center, Houston, PNP, purine nucleoside phosphorylase; t@, half-life of elimination; dNTP, deoxynucle TX 77030. otide triphosphate; CHO, Chinese hamster ovary; pol a, polymerase a. 1517 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. METABOUSM AND ACtiONS OF DIFLUORODEOXYGUANOSINE purchased from ICN Biochemicals (Costa Mesa, CA). All nucleosides were 0 purified by reverse-phase HPLC to greater than 99% purity. Cell CUltUre and Cytotoxicity. CHO cell lines were maintained in loga rithmic growth on 60-mm plastic dishes or roller bottles in McCoy's 5A H2N@5@> medium with 20% horse serum (GIBCO, Grand Island, NY). Under these conditions, the cell population-doubling time was 12 h. The cell line aC'@7, HO@ Ho@c::@ kindly supplied by Priscilla P. Saunders (20), is a CHO line deficient in dCyd kinase. In several experiments, cells were trypsinized, washed into calcium free McCoy's 5A medium, and grown in spinner flasks for 24 to 36 h before OH F use. Clonogenicity after drug treatment was determined by two methods. In the Deoxyguanoslrss Difluorodooxyguano first, cells were seeded on plates, treated as indicated, washed into fresh (dGuo) (dFdG) medium, and incubated for 5 days to allow colony formation. In the second procedure, cells were incubated with drugs in suspension culture. At the Fig. 1. Chemical structures of dGuo and dFdOuo (dFdG). indicated times portions of each culture were removed, washed into drug-free medium, plated in multiple dilutions at densities of 100—1000cells/plate,and incubated for 7—8days. Colonies were fixed and stained with methanol calculation assumes that nucleotides are uniformly distributed in total cell containing 0.5% crystal violet. The mean of at least 3 plates was determined; water. SD values were less than 10% of the mean in every case. Determinations ofdNTP. CHO cells incubated with 10 or 30 g@MdFdGuo Phosphorolysis of dFdGuo. The activity of PNP was determined by for 3 h were extracted by 60% methanol. The DNA polymerase assay as spectrophotometric assay. Normal substrates guanosine and inosine at 10, 20, modified