Umbach G E, Spitzer G, Ajani J A, Hug V, Thames H, Rudolph F B, Drewinko B
J Cancer Res Clin Oncol. 1986;111(3):273-6. doi: 10.1007/BF00389244.
The effect of drug concentration, exposure duration, and culture conditions on the cytotoxic activity of methotrexate (MTX) on normal granulocyte-macrophage colony-forming units in culture (GM-CFUC) was studied using a bilayer soft agar system with nucleoside-free medium. The degree of inhibition of colony formation depended on the type of serum supplementation. A 1 h or 2 h pulse treatment with 2 X 10(-4) M (100 micrograms/ml) MTX failed to kill GM-CFUC, when the cells were subsequently plated in a system containing 15% undialyzed fetal bovine serum (FBS). For continuous exposure the observed LD50 of MTX in the agar system was higher than 10(-4) M for 15% undialyzed FBS, 10(-5) M for 15% dialyzed FBS plus 0.25% undialyzed FBS, 10(-6) M for 15% dialyzed FBS, and 10(-8) M for 15% undialyzed horse serum. The difference for dialyzed FBS versus horse serum can be explained by differences in nucleoside concentrations. The difference for dialyzed FBS versus horse serum may be secondary to an enhancer of MTX in horse serum. For studying MTX sensitivity of human tumor cells in vitro, we suggest testing conditions that lie within the dose survival curve of GM-CFUC.
使用无核苷培养基的双层软琼脂系统，研究了药物浓度、暴露持续时间和培养条件对甲氨蝶呤（MTX）对培养的正常粒细胞-巨噬细胞集落形成单位（GM-CFUC）细胞毒性活性的影响。集落形成的抑制程度取决于血清补充的类型。当随后将细胞接种到含有15%未透析胎牛血清（FBS）的系统中时，用2×10⁻⁴ M（100微克/毫升）MTX进行1小时或2小时的脉冲处理未能杀死GM-CFUC。对于连续暴露，在琼脂系统中观察到的MTX的半数致死剂量（LD50）对于15%未透析FBS高于10⁻⁴ M，对于15%透析FBS加0.25%未透析FBS为10⁻⁵ M，对于15%透析FBS为10⁻⁶ M，对于15%未透析马血清为10⁻⁸ M。透析FBS与马血清之间的差异可以通过核苷浓度的差异来解释。透析FBS与马血清之间的差异可能继发于马血清中MTX的增强剂。为了在体外研究人肿瘤细胞对MTX的敏感性，我们建议测试处于GM-CFUC剂量存活曲线范围内的条件。
