Zhu Wei-Yong, Bunni M, Priest D G, DiCapua J L, Dressler J M, Chen Z, Melera Peter W
Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA.
J Exp Ther Oncol. 2002 Sep-Oct;2(5):264-77. doi: 10.1046/j.1359-4117.2002.01049.x.
DC-3F/FA3 cells (FA3) were derived from antifolate-sensitive CHL cells by selection for growth in folate-free media containing 15 pM [6S]-5CHOFH4. These cells undergo a 30-fold decrease in intracellular folates, overexpress folate receptor alpha (FR alpha) and metallothionein II, and display increased sensitivity to the dihydrofolate reductase (DHFR) targeted anti-folates methotrexate (MTX) and trimetrexate (TMTX), which can be attributed primarily to the folate pool status. Upon folate repletion by growth in 15 nM [6S]-5CHOFH4, they display a 5- and 10-fold increase in resistance to both drugs, respectively, even though folate pools are restored by only 43%. Enforced overexpression of FR alpha in transfectants cultured in nanomolar folate did not confer resistance to MTX but did support a modest 2-fold increase in resistance to TMTX. Enforced overexpression of MTII had a similar effect, but when both were overexpressed together no increase in resistance beyond that conferred by each one separately was noted, suggesting that both confer resistance to TMTX through a common downstream mechanism. Analysis of three independent low folate selected clones, FA3, FA7, and FA14, showed that each had a 5- to 6-fold increase in DHFR activity accompanied by a similar increase in DHFR protein level. However, no differences were detected in the DHFR gene copy number or in the steady-state amount of DHFR mRNA, suggesting that a posttranscriptional mechanism was responsible for the increase in DHFR expression.
DC - 3F/FA3细胞（FA3）源自对叶酸敏感的中国仓鼠肺细胞（CHL细胞），通过在含有15 pM [6S]-5 - 甲基四氢叶酸（[6S]-5CHOFH4）的无叶酸培养基中筛选生长而获得。这些细胞的细胞内叶酸含量下降30倍，叶酸受体α（FRα）和金属硫蛋白II过表达，并对靶向二氢叶酸还原酶（DHFR）的抗叶酸药物甲氨蝶呤（MTX）和三甲曲沙（TMTX）表现出更高的敏感性，这主要可归因于叶酸池状态。在15 nM [6S]-5CHOFH4中生长使叶酸得到补充后，它们对这两种药物的抗性分别增加了5倍和10倍，尽管叶酸池仅恢复了43%。在纳摩尔浓度叶酸培养的转染子中强制过表达FRα并未赋予对MTX的抗性，但确实使对TMTX的抗性适度增加了2倍。强制过表达金属硫蛋白II（MTII）有类似的效果，但当二者同时过表达时，并未观察到抗性增加超过各自单独过表达时的情况，这表明二者通过共同的下游机制赋予对TMTX的抗性。对三个独立的低叶酸筛选克隆FA3、FA7和FA14的分析表明，每个克隆的DHFR活性均增加了5至6倍，同时DHFR蛋白水平也有类似增加。然而，在DHFR基因拷贝数或DHFR mRNA的稳态量方面未检测到差异，这表明转录后机制导致了DHFR表达的增加。
