Flintoff Wayne F, Sadlish Heather, Gorlick Richard, Yang Rui, Williams Frederick M R
Department of Microbiology and Immunology, University of Western Ontario, Dental Sciences Bldg., Dock 15, London, Canada N6A 5C1.
Biochim Biophys Acta. 2004 Oct 14;1690(2):110-7. doi: 10.1016/j.bbadis.2004.05.008.
Osteosarcomas are common primary malignant bone tumors that do not respond to conventional low-dose treatments of methotrexate (Mtx), suggesting an intrinsic resistance to this drug. Previous work has shown that cDNAs generated from osteosarcoma mRNA from a fraction of patients contain sequence changes in the reduced folate carrier (RFC), the membrane protein transporter for Mtx. In this study, the functionality of the altered RFC proteins was assessed by fusing the green fluorescent protein (GFP) to the C-terminal, and examining the ability of the transfected constructs to complement a hamster cell line null for the carrier. Confocal microscopy and cell surface biotinylation indicated that all altered proteins were properly localized at the cell membrane. Only one of those examined, Leu291Pro, was unable to complement the null carrier line, but did bind Mtx at the cell surface. Thus, this alteration confers drug resistance since the carrier is unable to translocate the substrate across the cell membrane. Three alterations, Ser46Asn, Ser4Pro and Gly259Trp, while able to complement the carrier null line, conferred some degree of resistance to Mtx via a decreased rate of transport (Vmax). Another set of alterations, Glu21Lys, Ala7Val, and the combined changes Thr222Ile, Met254Thr, complemented the carrier null line and did not confer resistance to Mtx. Thus, some, but not all of these identified alterations in the RFC may contribute to the lack of responsiveness of osteosarcomas to Mtx treatment.
骨肉瘤是常见的原发性恶性骨肿瘤，对甲氨蝶呤（Mtx）的传统低剂量治疗无反应，提示对该药物存在内在抗性。先前的研究表明，部分骨肉瘤患者的mRNA产生的cDNA在还原型叶酸载体（RFC）（Mtx的膜蛋白转运体）中含有序列变化。在本研究中，通过将绿色荧光蛋白（GFP）融合到C末端来评估改变的RFC蛋白的功能，并检测转染构建体对缺乏该载体的仓鼠细胞系的互补能力。共聚焦显微镜和细胞表面生物素化表明，所有改变的蛋白都正确定位于细胞膜。在所检测的蛋白中，只有Leu291Pro不能互补缺乏载体的细胞系，但能在细胞表面结合Mtx。因此，这种改变导致耐药性，因为载体无法将底物转运穿过细胞膜。三种改变，Ser46Asn、Ser4Pro和Gly259Trp，虽然能够互补缺乏载体的细胞系，但通过降低转运速率（Vmax）赋予了一定程度的Mtx抗性。另一组改变，Glu21Lys、Ala7Val以及组合变化Thr222Ile、Met254Thr，互补了缺乏载体的细胞系，且未赋予Mtx抗性。因此，RFC中这些已鉴定的改变中，有些但并非全部可能导致骨肉瘤对Mtx治疗缺乏反应。
