Schuetz J D, Westin E H, Matherly L H, Pincus R, Swerdlow P S, Goldman I D
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298.
J Biol Chem. 1989 Sep 25;264(27):16261-7.
We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier. This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug. The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km. There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX. Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line. The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier. Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased. There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively. SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines. Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line. [3H]Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase. Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds. These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier. Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect.
我们报道了L1210白血病细胞系中膜蛋白的变化，该细胞系在甲氨蝶呤（MTX）-四氢叶酸辅助因子转运载体功能上存在高度特异性缺陷。这个克隆系MTXrA对MTX产生了100倍的抗性，是一步衍生而来的，并且在无药物的情况下经过120代仍表现出稳定的抗性。转运缺陷与流入Vmax降低10倍相关，而流入Km没有变化。MTXrA细胞系与亲代细胞系在MTX特异性细胞表面结合剂的水平或亲和力上没有差异，在用特异性亲和标记物——氚化MTX的N - 羟基琥珀酰亚胺酯处理后，44 kDa膜蛋白的标记也没有差异。与载体功能受损一致的是，在亲代细胞系中观察到的细胞内5 - 甲酰四氢叶酸对MTX流入的反式刺激在MTXrA细胞系中未得到证实。转运缺陷对MTX - 四氢叶酸辅助因子转运载体具有高度特异性。5 - 氟 - 2'-脱氧尿苷和2 - 脱氧葡萄糖的初始摄取率未改变，事实上，α - 氨基异丁酸的流入和净转运增加。该细胞系对分别利用特定氨基酸和核苷转运载体的苯丙氨酸氮芥或阿糖胞苷没有交叉抗性。用考马斯亮蓝染色的纯化质膜制剂的SDS - 聚丙烯酰胺凝胶电泳显示亲代细胞系和MTXrA细胞系之间存在几种蛋白质差异。最显著的是一条约190 kDa的条带，其迁移率略高于亲代细胞系中一条染色较浅的条带。用[³H]硼氢化钠对细胞进行标记也在MTXrA细胞系中鉴定出一个约190 kDa的独特蛋白峰，在用神经氨酸酶预先处理细胞后该峰消失。与多药耐药基因相关的蛋白质或mRNA的缺失以及对柔红霉素或三甲曲沙缺乏交叉抗性表明，这种对MTX的耐药机制与高分子量杂环化合物所观察到的多药耐药现象完全无关。这些数据首次证明了在一个对MTX具有明显独特转运缺陷的高度抗性L1210小鼠白血病细胞系中存在膜蛋白差异，这种缺陷似乎与四氢叶酸辅助因子载体的迁移受损有关。现在需要进一步研究以阐明这些蛋白质中的一种或多种在转运缺陷中可能的作用。
