title: Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines.
abstract: Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTX(R) ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFCI RNA levels by a quantitative RT-PCR assay. RFCI RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTX(R) ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines.
authors.affiliation: Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892, USA. moscowj@pbmac.nci.nih.gov
authors.forename: J A
authors.initials: JA
authors.lastname: Moscow
authors.forename: T
authors.initials: T
authors.lastname: Connolly
authors.forename: T G
authors.initials: TG
authors.lastname: Myers
authors.forename: C C
authors.initials: CC
authors.lastname: Cheng
authors.forename: K
authors.initials: K
authors.lastname: Paull
authors.forename: K H
authors.initials: KH
authors.lastname: Cowan
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005492:Folic Acid / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000494:pharmacology*; D009369:Neoplasms / Q000188:drug therapy*; D012333:RNA, Messenger / Q000032:analysis; D058978:Reduced Folate Carrier Protein; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D005492:Folic Acid; D008727:Methotrexate