Regulation of folate-binding protein gene expression by DNA methylation in methotrexate-resistant KB cells

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.