Hsueh C T, Dolnick B J
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263.
Biochem Pharmacol. 1994 Mar 15;47(6):1019-27. doi: 10.1016/0006-2952(94)90413-8.
Folate-binding protein (FBP) is responsible for the cellular transport of folate and methotrexate (MTX) in human KB (nasopharyngeal epidermoid carcinoma) cells. The levels of membrane-associated FBP and FBP mRNA are decreased 70-80% in an MTX-resistant KB subline (KB1BT) (Hsueh C-T and Dolnick BJ, Oncol Res 4: 497-505, 1992). Southern blot analysis did not reveal any differences in FBP gene organization or copy number between KB1BT and KB cells. However, there was a 70% decrease in the FBP gene transcription rate and no change in FBP mRNA stability in KB1BT cells. Assessing genomic DNA methylation by MspI and HpaII restriction analysis suggested that the FBP gene in KB1BT cells was more methylated than in KB cells. These alterations in the expression, transcription rate and DNA methylation state of the FBP gene did not change when KB1BT cells were grown in the absence of MTX for 8 months (MTX-free KB1BT). When MTX-free KB1BT cells were exposed to 2.5 microM 5-aza-2'-deoxycytidine for 72 hr, the FBP gene became hypomethylated and the levels of membrane-associated FBP and FBP mRNA increased by 2- to 3-fold. These data indicate that decreased FBP gene expression in KB1BT cells results from increased DNA methylation.
叶酸结合蛋白（FBP）负责叶酸和甲氨蝶呤（MTX）在人KB（鼻咽表皮样癌）细胞中的细胞转运。在耐MTX的KB亚系（KB1BT）中，膜相关FBP和FBP mRNA水平降低了70 - 80%（Hsueh C-T和Dolnick BJ，《肿瘤研究》4：497 - 505，1992）。Southern印迹分析未揭示KB1BT和KB细胞之间FBP基因组织或拷贝数的任何差异。然而，KB1BT细胞中FBP基因转录率降低了70%，FBP mRNA稳定性没有变化。通过MspI和HpaII限制性分析评估基因组DNA甲基化表明，KB1BT细胞中的FBP基因比KB细胞中的甲基化程度更高。当KB1BT细胞在无MTX的情况下培养8个月（无MTX的KB1BT）时，FBP基因在表达、转录率和DNA甲基化状态方面的这些改变并未改变。当无MTX的KB1BT细胞暴露于2.5 microM 5-氮杂-2'-脱氧胞苷72小时时，FBP基因去甲基化，膜相关FBP和FBP mRNA水平增加了2至3倍。这些数据表明，KB1BT细胞中FBP基因表达降低是由于DNA甲基化增加所致。
