Gene amplification and increased expression of the reduced folate carrier in transport elevated K562 cells.
Gene amplification and increased expression of the reduced folate carrier in transport elevated K562 cells.
复制标题
在运输升高的 K562 细胞中基因扩增和还原叶酸载体的表达增加。
DOI：
10.1016/s0006-2952(97)00639-4
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发表时间：
1998
影响因子：
5.8
通讯作者：
Matherly,LH
中科院分区：
文献类型：
--
作者：
Wong,SC;Zhang,L;Proefke,SA;Hukku,B;Matherly,LH
K562.4CF细胞的甲氨蝶呤转运能力提高6倍的分子基础（Matherly等人，Cancer Res. 51：3420-3426，1991）用还原叶酸载体（RFC）cDNA、基因组和抗体探针进行研究。Southern分析表明，RFC基因拷贝增加（104 - 5倍）在K562.4CF野生型K562细胞。使用基因组RFC探针的荧光原位杂交证实了RFC基因定位于21号染色体的q臂。在K562.4CF细胞中，21号染色体（61%的中期分裂相）正常拷贝的频繁丢失伴随着RFC基因扩增和扩增的RFC基因片段易位到几个（2至6个）不同的染色体位点，在野生型细胞中未见。特别强烈的RFC信号被映射到染色体2和15中的均匀染色区域。RFC基因拷贝数的增加伴随着北方印迹法测得的主要3.1 kb RFC转录物的类似增加，以及用RFC C-末端肽抗体探测的蛋白质印迹法测得的广泛迁移（80-95 kDa）RFC蛋白水平的107倍升高。这些结果表明，选择具有生长限制浓度的还原叶酸（0.4 nM亚叶酸）的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。
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 (≈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 ≈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.