Department of Cancer Medicine, Blackburn Building D06, The University of Sydney, Sydney, NSW 2006, Australia.
Cancer Chemother Pharmacol. 2010 May;66(1):121-7. doi: 10.1007/s00280-009-1142-2. Epub 2009 Sep 27.
Nucleoside and base modulation of the cytotoxicity of nucleic acid and folate antimetabolite drugs has been widely discussed. Many investigators have observed reduced toxicity due to circumvention of drug-induced inhibition of de novo purine and pyrimidine synthesis. However, exogenous purine nucleosides and bases may also enhance the cytotoxicity of even moderate concentrations of antifolate drugs (MTX and PTX) which inhibit dihydrofolate reductase. In this study, the effects of nucleosides in the medium on the cytotoxicity and deoxyribonucleoside triphosphate pools after brief exposure of cultured cells to methotrexate have been studied in cultured L1210 murine leukaemia cells.
Cell viability was determined by trypan blue exclusion assay. Colony formation was assessed by microtitration cloning assay. The deoxyribonucleotides were measured by a modification of the DNA polymerase assay. Purines were extracted with trioctylamine and 1,1,2-trichlorotrifluoroethane buffer and concentrations of purine bases were determined by HPLC.
Subculture of drug-treated cells in fresh medium containing 10% FCS led to greater toxicity than sub culture in 'conditioned' medium, i.e. fresh medium in which logarithmically growing cells had been cultured for 24 h before separation. Cells resuspended in fresh medium had increased dATP and sustained inhibition of dTTP levels, while cells subcultured in 'conditioned' medium had no elevation of dATP. Hypoxanthine concentration determined by HPLC in 'conditioned' medium was 0.9 microM compared to 6.7 microM in fresh medium. Resuspension of drug-treated cells in conditioned medium supplemented with 10 or 100 microM HX enhanced cytotoxicity and increased the dATP levels.
These results add further evidence that purines present in normal culture conditions are important determinants of methotrexate cytotoxicity. Elevation of dATP levels after methotrexate treatment is an important modulator of cytotoxicity.
核苷和碱基对核酸和叶酸抗代谢物药物的细胞毒性的调节作用已被广泛讨论。许多研究人员观察到，由于药物诱导的从头嘌呤和嘧啶合成抑制作用的规避，毒性降低。然而，外源性嘌呤核苷和碱基也可能增强甚至中等浓度的抗叶酸药物（MTX 和 PTX）的细胞毒性，这些药物抑制二氢叶酸还原酶。在这项研究中，在短暂暴露于甲氨蝶呤后，研究了培养基中的核苷对培养的 L1210 白血病细胞中甲氨蝶呤细胞毒性和脱氧核苷三磷酸池的影响。
通过台盼蓝排除试验测定细胞活力。通过微量滴定克隆测定法评估集落形成。脱氧核苷酸通过 DNA 聚合酶测定法的改进来测量。嘌呤用三辛胺和 1,1,2-三氯三氟乙烷缓冲液提取，并用 HPLC 测定嘌呤碱基的浓度。
在含有 10%FCS 的新鲜培养基中培养药物处理的细胞比在“条件”培养基中培养毒性更大，即对数生长期细胞在分离前在新鲜培养基中培养 24 小时。悬浮在新鲜培养基中的细胞 dATP 增加，并持续抑制 dTTP 水平，而在“条件”培养基中培养的细胞则没有 dATP 升高。通过 HPLC 确定“条件”培养基中次黄嘌呤的浓度为 0.9µM，而新鲜培养基中为 6.7µM。用 10 或 100µM HX 补充的“条件”培养基悬浮药物处理的细胞可增强细胞毒性并增加 dATP 水平。
这些结果进一步证明，正常培养条件下存在的嘌呤是甲氨蝶呤细胞毒性的重要决定因素。甲氨蝶呤处理后 dATP 水平的升高是细胞毒性的重要调节剂。
