Wong S C, Zhang L, Proefke S A, Hukku B, Matherly L H
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201, USA.
Biochem Pharmacol. 1998 Apr 1;55(7):1135-8. doi: 10.1016/s0006-2952(97)00639-4.
The molecular bases for the 6-fold elevated methotrexate transport capacity of K562.4CF cells (Matherly et al., Cancer Res. 51: 3420-3426, 1991) were studied with reduced folate carrier (RFC) cDNA, genomic, and antibody probes. Southern analysis showed that RFC gene copies were increased (approximately 4- to 5-fold) in K562.4CF over wild-type K562 cells. Fluorescence in situ hybridization using a genomic RFC probe confirmed the localization of the RFC gene to the q-arm of chromosome 21. In K562.4CF cells, the frequent loss of a normal copy of chromosome 21 (61% of metaphases) was accompanied by RFC gene amplification and translocations of amplified RFC gene fragments to several (2 to 6) different chromosomal loci not seen in wild-type cells. Particularly intense RFC signals were mapped to homogeneously staining regions in chromosomes 2 and 15. Increased RFC gene copies were accompanied by a similar increase in the major 3.1 kb RFC transcript by northern blotting and an approximately 7-fold elevated level of the broadly migrating (80-95 kDa) RFC protein on a western blot probed with an RFC C-terminal peptide antibody. These results demonstrate that selection of cells with a growth-limiting concentration of reduced folates (0.4 nM of leucovorin) is sufficient to promote chromosomal aberrations, including gene amplification and translocations that result in increased RFC expression and folate transport.
利用还原型叶酸载体（RFC）的互补DNA、基因组及抗体探针，对K562.4CF细胞（Matherly等人，《癌症研究》51: 3420 - 3426, 1991）中氨甲蝶呤转运能力提高6倍的分子基础进行了研究。Southern分析显示，与野生型K562细胞相比，K562.4CF细胞中的RFC基因拷贝数增加（约4至5倍）。使用基因组RFC探针进行的荧光原位杂交证实，RFC基因定位于21号染色体的长臂。在K562.4CF细胞中，21号染色体正常拷贝的频繁丢失（61%的中期细胞）伴随着RFC基因扩增以及扩增的RFC基因片段易位至几个（2至6个）野生型细胞中未见的不同染色体位点。特别强烈的RFC信号定位于染色体2和15的均匀染色区。通过Northern印迹法检测到，RFC基因拷贝数增加伴随着主要的3.1 kb RFC转录本有类似增加，在用RFC C末端肽抗体进行Western印迹检测时，广泛迁移的（80 - 95 kDa）RFC蛋白水平升高了约7倍。这些结果表明，用生长限制浓度的还原型叶酸（0.4 nM亚叶酸钙）选择细胞足以促进染色体畸变，包括导致RFC表达和叶酸转运增加的基因扩增和易位。
