Moscow J A, Connolly T, Myers T G, Cheng C C, Paull K, Cowan K H
Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892, USA.
Int J Cancer. 1997 Jul 3;72(1):184-90. doi: 10.1002/(sici)1097-0215(19970703)72:1<184::aid-ijc26>3.0.co;2-i.
Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTX(R) ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFCI RNA levels by a quantitative RT-PCR assay. RFCI RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTX(R) ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines.
在几种因对甲氨蝶呤（MTX）耐药而筛选出的细胞系中，已观察到由于还原型叶酸载体（RFC）活性降低导致的甲氨蝶呤转运缺陷。然而，由于MTX耐药是多因素的，所以很难量化所选细胞系中RFC活性变化的相对重要性，更难以确定天然存在的RFC活性对未选细胞系MTX敏感性的相对贡献。我们通过研究转染了人RFC基因RFC1的转运缺陷细胞系，并将RFC1相对表达与NCI抗癌药物筛选中使用的一组细胞系中甲氨蝶呤和三甲曲沙（TMTX）的生长抑制（GI50）相关联，来研究RFC在MTX耐药中的作用。转染RFC1的转运缺陷、MTX耐药的ZR-75-1人乳腺癌细胞（MTX(R) ZR-75-1）克隆对MTX的敏感性比对照细胞克隆高250倍，对TMTX的耐药性比对照细胞克隆高300倍，这表明RFC活性的恢复对MTX和TMTX的细胞毒性有显著影响。我们还通过定量RT-PCR测定法对NCI药物筛选面板中的60个细胞系中的40个进行了RFC1 RNA水平的检测。RFC1 RNA水平在15倍的范围内变化，仅发现1个细胞系表达缺失。使用6天药物暴露试验的数据，RFC1与MTX呈正相关，与TMTX细胞毒性呈负相关。正如转染研究所预测的，计算出的MTX和TMTX效力差异与细胞系的RFC1 RNA水平的相关性更强。此外，还检测了NCI抗癌药物筛选数据库中细胞毒性谱与RFC1 RNA水平或计算出的MTX-TMTX效力差异相关的化合物在转染RFC1的MTX(R) ZR-75-1细胞系中的MTX摄取抑制和细胞毒性。总体而言，我们的数据证明了RFC1在MTX耐药中作为转基因以及在未选细胞系中作为组成性表达基因的重要性。
