Masters J, Keeley B, Gay H, Attardi G
Mol Cell Biol. 1982 May;2(5):498-507. doi: 10.1128/mcb.2.5.498-507.1982.
Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.
通过将人细胞系HeLa BU25和VA2 - B暴露于逐渐增加浓度的药物中，已从这些细胞系中分离出几种对1.8×10⁻⁴ M DL - 甲氨蝶呤（MTX）具有抗性的变体。在不同变体之间观察到了显著的表型和染色体组成变异性。所有抗性细胞系均表现出二氢叶酸还原酶（DHFR）活性和DHFR含量大幅增加；然而，不同变体之间的DHFR活性水平差异很大，范围在亲本水平的约35至275倍之间。在没有选择压力的情况下，增加的DHFR活性不稳定，除了一个细胞系外，所有细胞系在25至200天的不同时间段内DHFR活性完全丧失。MTX抗性细胞系在染色体组成上表现出异常，这包括一些变体的大多数细胞中出现一组重复的染色体，以及所有细胞系中均存在双微体染色体。在没有MTX的情况下，对双微体染色体丢失与DHFR活性丧失之间相关性的分析结果与双微体染色体包含扩增的DHFR基因这一观点一致。然而，最显著的发现是，与小鼠系统中所报道的情况相反，在不同变体中可识别的双微体染色体数量差异很大，与DHFR活性水平或在没有选择压力时MTX抗性的不稳定程度均无任何关系。这些以及其他观察结果表明，在人类MTX抗性变体中存在另一组DHFR基因，其在总数中占不同比例，与正常染色体相关，并且在没有选择压力时可能不稳定。
