The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It Methotrexate (MTX) is an analog of dihydrofolic acid, which strongly binds to dihydrofolate reductase, thereby producing a depletion of reduced folates, which are essential for the de novo synthesis of thymidilate and purines. The mechanism of cell death by MTX varies between different cell types, since it has been reported that in some cells the induced shortage of thymidilate is the predominant cause of cell death, whereas in others the purineless state appears to be the predominant mechanism (1-3). The only consistently reported change of deoxyribonucleoside triphosphates pool induced by MTX is that of TTP, which diminishes both in vivo (4) and in vitro (5). In a previous study on the effect of MTX on human osteosarcoma (6), it was suggested that osteosarcoma cells formed their TTP pool mainly through the contribution of the de novo pathway, and it was proposed that an activation of the deoxyribonucleosides salvage pathway in tumor cells could be a mechanism of resistance to MTX in addition to those already reported. There are many evidences that the effect of MTX on tumor cells may be circumvented by the presence of dThd and hypoxanthine (Hyp) in the cell medium (7,8). Therefore, it was reasoned that a combination of MTX with agents that interfere with the salvage pathway could block this reversion and favor the cyto- Deoxyribonucleoside Incorporation into DNA. After being counted, tumor cells were centrifuged at 500 x g for 10 min, washed once with Eagle's minimal essential medium (ME medium), and resuspended in ME medium at 1 x 106 cells per ml. After being submitted to different experimental conditions, 1-ml aliquots received pulses (30 min at 37°C) with 0.2 ,uCi (1 Ci = 37 GBq) of [3H]dCyd (specific activity, 20-40 Ci/mmol), 2 uCi of [3H]dThd (specific activity, 20 Ci/mmol), or 2 ,uCi of [3H]dUrd (specific activity, [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] Ci/mmol). The pulses were terminated by the addition of 0.1 ml of 0.1 M PPj, 0.2 ml of DNA carrier (2.5 mg/ml), 0.2 ml of H20, and 0.25 ml of 12.5% perchloric acid. The tubes were centrifuged at 3000 x g for 10 min, and the precipitates were resuspended in 0.5 ml of 0.2 M NaOH. After addition of 1 ml of H20 and 0.25 ml of 12.5% perchloric acid, the tubes were recentrifuged, and the procedure was repeated once more. The precipitate was resuspended in 0.3 ml of Soluene-350 (Packard), and radioactivity was measured with 7 ml of toluene-Omnifluor in a Beckman LS 8100 scintillation counter.Determination of TTP Pools. The technique used was a modification of that described by Kinahan et al. (11). The cells were removed by paracentesis, washed once with ME medium, and resuspended at 1 x 106 cells per ml. After incubation for 2 hr at 37°C under different conditions, 40 x 106 cells were centrifuged, resuspended in 1 ml of 60% methanol, and stored overnight at -200C. The next morning t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.