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Triphosphate Accumulation, DNA Damage, and Growth Inhibition Following Exposure to CB3717 and Dipyridamole Nicola J

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Triphosphate Accumulation, DNA Damage, and Growth Inhibition Following Exposure to CB3717 and Dipyridamole Nicola J (CANCER RESEARCH 51. 2346-2352, May I. 1991) Mechanism of Cell Death following Thymidylate Synthase Inhibition: 2'-Deoxyuridine-5'-triphosphate Accumulation, DNA Damage, and Growth Inhibition following Exposure to CB3717 and Dipyridamole Nicola J. Curtin,1 Adrian L. Harris, and G. Wynne Aherne Cancer Research L'nil, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne [N. J. C.J; Imperial Cancer Research Fund Clinical Oncology Unit, Churchill Hospital, Headington, Oxon ¡A.L. H.]; Department of Biochemistry, University of Surrey, Guildford, Surrey [G. W. A.], England ABSTRACT but one hypothesis, based on the study of bacterial mutants (3- The thymidylate synthasc inhibitor /V'°-propargyl-5,8-dideazafolic 5), is that TS inhibition leads not only to a reduction in dTTP levels but also, as dUMP accumulates behind the block, to the acid (CB3717) inhibits the growth of human lung carcinoma A549 cells. formation of dUTP. The levels of dUTP eventually overwhelm The cytotoxicity of CB3717 is potentiated by the nucleoside transport inhibitor dipyridamole (DP), which not only inhibits the uptake and dUTPase (the enzyme which breaks down dUTP to dUMP) therefore salvage of thymidine but also inhibits the efflux of deoxyuridine, and the levels of dUTP increase. DNA polymerase can utilize thereby enhancing the intracellular accumulation of deoxyuridine nucleo- dUTP and dTTP with equal efficiency (6), such that uracil is tides. Measurement of intracellular deoxyuridine triphosphate (dUTP) misincorporated into DNA. Uracil in DNA is excised rapidly pools, by sensitive radioimmunoassay, demonstrated a large increase in by uracil glycosylase, leaving an apyrimidinic site. During repair response to CB3717, in a dose- and time-related manner, and this of apyrimidinic sites, in the presence of unbalanced dUTP/ accumulation was enhanced by coincubation with DP. In untreated cells dTTP ratios, uracil is likely to be reinserted, causing a futile and those treated with DP alone, dUTP was close to or below the limit cycle of excision, repair, and reinsertion, leading to DNA strand of detection of the assay. In cells treated for 24 h with 3 MMCB3717 breakage and ultimately cell death. Whether or not thymineless (concentration producing 50% growth inhibition) the intracellular dUTP was 46.1 ±9.6 (SEM) pumi III" cells and after 24 h exposure to 30 n\i death occurs by this mechanism in mammalian cells has not CB3717, 337.5 ±37.9 pmol dUTP/106 cells was detected. There was been fully determined; some investigators have presented evi significant enhancement by DP of the accumulation of ill 11*in cells dence supporting this theory (7-9), whereas others have pre treated with CB3717; coincubation of cells with 1 MMDP + 3 MMCB3717 sented evidence to the contrary (10, 11). for 24 h resulted in intracellular dLTP levels of 174.7 ±57.7 pmol/10" In order to investigate the role of dUTP in thymineless death cells. Accumulation of DNA strand breaks, measured by alkaline elution, in mammalian cells we have studied the combined effect of also increased in response to CB3717 concentration and exposure period. CB3717 and the nucleoside transport inhibitor, DP on dUTP Newly synthesized (nascent) DNA was more sensitive to damage by pools in relation to DNA damage and cytotoxicity. Salvage of CB3717 than was mature DNA. As with the accumulation of dUTP, dThd is a mechanism by which cells may bypass TS inhibition. coincubation with DP also enhanced the accumulation of strand breaks, By inhibiting dThd uptake, DP potentiated CB3717 toxicity; whereas DP alone had little or no effect on DNA fragmentation. however, this potentiation was greater than that achieved by When data for cells treated with CB3717 alone and CB3717 in limiting dThd availability with dialyzed serum (12). This dis combination with DP were combined, there was a significant correlation crepancy could be explained by the observation that DP also of intracellular dUTP levels with the level of DNA strand breaks. This inhibited deoxyuridine efflux and we postulated that DP might strongly suggests that growth inhibition following thymidylate synthase inhibition is mediated through an increase in intracellular dill', leading therefore help to maintain high deoxyuridine nucleotide pools to uracil misincorporation into DNA, its subsequent excision, and result and that this was responsible for its greater potentiating effect ant strand breakage. than dialyzed serum (12). Indeed, by inhibiting deoxyuridine efflux, DP has been shown to increase intracellular dUMP levels in fluorouracil-treated cells (13). INTRODUCTION To confirm our hypothesis that DP was potentiating CB3717 TS2 is a key enzyme of DNA synthesis and therefore a prime cytotoxicity in part by promoting dUTP accumulation, it was target for cancer chemotherapy. Methotrexate and fluorodeox- necessary to measure dUTP levels in cells treated with CB3717 yuridine can both give rise to TS inhibition but attempts to alone and in combination with DP. The standard methods for determine their mechanism of cytotoxicity are complicated by quantification of deoxynucleotide triphosphates are high per the fact that they also have other sites of action; methotrexate formance liquid chromatography and the indirect DNA polym also inhibits purine biosynthesis and fluorodeoxyuridine may erase assay. Both methods are unsuitable for dUTP determi be incorporated into DNA. In contrast the antifolate drug nation: high performance liquid chromatography because the method lacks sensitivity and hence large numbers of cells are CB3717 specifically inhibits TS (1, 2) and this makes it an required, while the DNA polymerase assay is very sensitive but excellent tool for the study of thymineless death in mammalian does not distinguish between dUTP and dTTP (6). In view of cells. these problems, we have developed a highly sensitive radio The mechanism of thymineless death has not been defined immunoassay method capable of detecting 3.78 fmol dUTP Received 6/11/90: accepted 2/13/91. (14). This has allowed the detection of small changes in dUTP The costs of publication of this article were defrayed in part by the payment levels using low numbers of cells (IO6) treated with biologically of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. relevant doses of CB3717 and DP. ' This work was supported by the North of England Cancer Research Cam We report here a study of the effects of CB3717 and DP on paign (N. J. C. and A. L. H.) and the Cancer Research Campaign of Great Britain dUTP pools in parallel with determinations of DNA damage (G. W. A.). A preliminary report of this work was presented at the Annual General Meeting of the British Association for Cancer Research, March 1990 (Br. J. by alkaline elution and cytotoxicity. The results show that Cancer, 62: 508. 1990). 2The abbreviations used are: TS. thymidylate synthase: DP, dipyridamole; intracellular dUTP accumulation is directly proportional to dThd. thymidine; CB3717. A'lo-propargyl-5,8-dideazafolic acid; IC50, concentra DNA damage and related to growth inhibition of human lung tion producing 50% growth inhibition; dNTP, deoxynucleoside triphosphate. carcinoma A549 cells treated with CB3717 and DP. 2346 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. CELL DEATH AFTER TS INHIBITION MATERIALS AND METHODS cells were exposed to fresh control medium or that containing CB3717 and/or DP. After the desired incubation period, the medium was Cell Culture. Human lung carcinoma A549 cells (15, 16) were grown removed by aspiration, and the dish was placed on ice and flooded with in 6-well cluster dishes (Falcon; Becton Dickinson, Oxford, England) 1 ml ice-cold 0.4 M perchloric acid. The cells were removed from each in RPMI 1640 (Northumbria Biologicals, Cramlington, England) sup well with a cell scraper and the suspensions were transferred to chilled plemented with 10% fetal bovine serum (Biological Industries, Glasgow, centrifuge tubes, vigorously vortex mixed, and placed on ice for 30 min. Scotland), 500 ID/ml penicillin, and 500 Mg/ml streptomycin (Gibco, They were centrifuged for 20 min at 1500 x g at 4°Candthe supernatant Paisley, Scotland) at 37°Cin an atmosphere of 5% CO? in air. For was carefully decanted. The volume was measured and neutralized with growth inhibition assays, cells were seeded at 2 x IO4cells/well in 2 ml 0.5 volumes ice-cold 0.72 M KOH in 0.16 M KHCO3. Alter vortex medium. The following day, the medium was replaced with fresh control mixing, the extracts were kept on ice for 1 h and centrifuged as before. or experimental medium containing CB3717 and/or DP. After 72 h The supernatant fluid was transferred to duplicate vials and stored in cells were trypsinized and counted on a Coulter Counter (Coulter liquid nitrogen until assayed for dUTP levels. Replicate wells were used Electronics, Luton, England). for cell counts. Alkaline Elution. The method for the detection of DNA strand breaks Intracellular dUTP pools were measured using a previously described by alkaline elution has been reviewed in detail (15). The technique radioimmunoassay procedure with a sensitivity of 3.78 fmol (14). utilizes filters which mechanically impede the passage of long DNA Briefly, cell extracts were treated with 0.5 Msodium periodate to remove strands. Short DNA strands will be eluted rapidly from the filters and interfering ribonucleotides and an aliquot (50-500 //I. depending on larger DNA strands will be retained on the filter for longer periods. the degree of exposure of the cells to CB3717) was subjected to Experimental cells exposed to varying drug concentrations are coeluted chromatography on QAE A25 Sephadex (Pharmacia, Milton Keynes, with internal standard cells, which have been irradiated immediately England). Following elution of dUMP and dUDP with 0.3 M KH2PO4, prior to elution, in alkaline buffer. Measurements of the fraction of dUTP was eluted with 0.7 M KH2PO4 and the eluate collected in 0.8- DNA from the experimental cells remaining on the filter, compared ml fractions.
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