Taylor I W, Tattersall M H
Cancer Res. 1981 Apr;41(4):1549-58.
Methotrexate (MTX)(2 x 10(-8) M) inhibited DNA synthesis in CCRF-CEM cells, causing cells to accumulate in early S phase while cellular RNA content and cell size continued to increase. Two-parameter flow cytometric analysis of DNA and RNA showed these cells to be unbalanced with excessive RNA relative to DNA content. Fifty % of cells remained viable after a 96-hr exposure to 2 x 10(-8) M MTX. In contrast, 10(-4) M MTX inhibited cell cycle progression of cells in both G1 and S phases and also prevented the development of unbalanced growth. In this instance, cell viability was reduced to 10% after 96 hr of drug exposure. The relative contribution of inhibition of thymidylate and purine biosynthesis to MTX cytotoxicity was investigated by addition of exogenous thymidine (10(-5) M) or hypoxanthine (10(-4) M). Thymidine reduced the cytotoxicity and inhibition of DNA synthesis caused by both doses of MTX and prevented classical unbalanced growth with 2 x 10(-8) m MTX; treatment with 10(-4) M MTX resulted in a form of unbalanced growth where cells had a relative excess of DNA compared with RNA content. The addition of hypoxanthine enhanced the classical unbalanced growth pattern seen with 2 x 10(-8) M MTX but was accompanied by a partial reduction of both the MTX-induced cytotoxicity and the inhibition of DNA synthesis (to an extent similar to that seen with exogenous thymidine). Potentiation of cell killing was observed with the addition of hypoxanthine to cells treated with 10(-4) M MTX. Complete rescue from MTX cytotoxicity at both concentrations was found only when both thymidine and hypoxanthine were present. These finding suggest that MTX cytotoxicity is associated with inhibition of DNA synthesis resulting from the disturbance of both thymidylate and purine biosynthesis.
甲氨蝶呤（MTX）（2×10⁻⁸ M）抑制CCRF - CEM细胞中的DNA合成，使细胞积聚在S期早期，而细胞RNA含量和细胞大小持续增加。对DNA和RNA进行双参数流式细胞术分析显示，这些细胞不平衡，RNA相对于DNA含量过多。暴露于2×10⁻⁸ M MTX 96小时后，50%的细胞仍存活。相比之下，10⁻⁴ M MTX抑制G1期和S期细胞的细胞周期进程，并阻止不平衡生长的发展。在这种情况下，药物暴露96小时后细胞活力降至10%。通过添加外源性胸苷（10⁻⁵ M）或次黄嘌呤（10⁻⁴ M）研究了胸苷酸和嘌呤生物合成抑制对MTX细胞毒性的相对贡献。胸苷降低了两种剂量MTX引起的细胞毒性和DNA合成抑制，并防止了2×10⁻⁸ m MTX引起的典型不平衡生长；用10⁻⁴ M MTX处理导致一种不平衡生长形式，即细胞的DNA相对于RNA含量相对过量。添加次黄嘌呤增强了2×10⁻⁸ M MTX所见的典型不平衡生长模式，但同时MTX诱导的细胞毒性和DNA合成抑制均部分降低（降低程度与外源性胸苷所见相似）。在10⁻⁴ M MTX处理的细胞中添加次黄嘌呤观察到细胞杀伤增强。仅当胸苷和次黄嘌呤都存在时，才发现两种浓度的MTX细胞毒性都能完全解除。这些发现表明，MTX细胞毒性与胸苷酸和嘌呤生物合成紊乱导致的DNA合成抑制有关。
