Miyachi H, Takemura Y, Kobayashi H, Ando Y
Department of Clinical Pathology, Tokai University School of Medicine, Kanagawa, Japan.
Cancer Lett. 1995 Jan 6;88(1):93-9. doi: 10.1016/0304-3835(94)03616-q.
Various alterations of the dihydrofolate reductase (DHFR) gene are involved in resistance. In order to understand the mechanism that induce such gene alterations in human leukemia cells, we studied the expression products of DHFR gene in trimetrexate (TMQ)- and/or methotrexate (MTX)-resistant sublines derived from a MOLT-3 human leukemia cell line. A 200-fold TMQ-resistant subline (MOLT-3/TMQ200) expressed the mutated DHFR mRNA, with a base change (T-->C) at the second position of codon 31, as well as the wild type gene. A MTX-resistant subline derived from MOLT-3/TMQ200 (MOLT-3/TMQ200-MTX500) showed a further increase in the expression of the mutated DHFR mRNA, compared to MOLT-3/TMQ200, with a marked decrease of expression of the wild type DHFR mRNA, which is confirmation of amplification of the mutated DHFR gene. By contrast, a 10,000-fold MTX-resistant subline (MOLT-3/MTX10,000) over-expressed the wild type DHFR mRNA, which is confirmation of amplification of the wild type gene. Increased levels of the DHFR enzyme in these sublines were proportional to expression levels of the DHFR mRNA. The DHFR enzyme expressed in MOLT-3/TMQ200-MTX500 cells showed a 40-fold increase in the Ki values for both MTX and TMQ, compared with values for the wild type DHFR expressed in both MOLT-3/MTX10,000 and its parent cell line. These findings suggest that the altered DHFR gene, which was introduced in MOLT-3 cells by exposure to TMQ, gave rise to a variant enzyme with reduced affinity to antifolates, and that complex DHFR alterations confer drug-resistant phenotypes in antifolate-resistance. Structural difference between the antifolates could be important in the introduction of the differential DHFR gene alterations in the antifolate resistance.
二氢叶酸还原酶（DHFR）基因的各种改变与耐药性有关。为了了解在人白血病细胞中诱导此类基因改变的机制，我们研究了源自MOLT - 3人白血病细胞系的三甲曲沙（TMQ）和/或甲氨蝶呤（MTX）耐药亚系中DHFR基因的表达产物。一个对TMQ耐药200倍的亚系（MOLT - 3/TMQ200）表达了突变的DHFR mRNA，在密码子31的第二个位置有一个碱基变化（T→C），同时也表达野生型基因。从MOLT - 3/TMQ200衍生出的一个对MTX耐药的亚系（MOLT - 3/TMQ200 - MTX500）与MOLT - 3/TMQ200相比，突变的DHFR mRNA表达进一步增加，而野生型DHFR mRNA的表达显著下降，这证实了突变的DHFR基因发生了扩增。相比之下，一个对MTX耐药10000倍的亚系（MOLT - 3/MTX10000）过度表达野生型DHFR mRNA，这证实了野生型基因的扩增。这些亚系中DHFR酶水平的增加与DHFR mRNA 的表达水平成正比。与在MOLT - 3/MTX10000及其亲本细胞系中表达的野生型DHFR相比，在MOLT - 3/TMQ200 - MTX500细胞中表达的DHFR酶对MTX和TMQ的Ki值增加了40倍。这些发现表明，通过暴露于TMQ在MOLT - 3细胞中引入的改变的DHFR基因产生了一种对抗叶酸亲和力降低的变体酶，并且复杂的DHFR改变在抗叶酸耐药性中赋予了耐药表型。抗叶酸之间的结构差异可能在抗叶酸耐药性中引入不同的DHFR基因改变方面很重要。
