REDUCED FOLATE CARRIER GENE (RFC1) EXPRESSION AND ANTIFOLATE RESISTANCE IN TRANSFECTED AND NONSELECTED CELL-LINES

Methotrexate transport deficiency due to decreased reduced folate carr ier (RFC) activity has been observed in several cell lines selected fo r resistance to methotrexate (MTX). Since MTX resistance is multifacto rial, however, it is difficult to quantify the relative Importance of changes in RFC activity in selected cell lines and even more so to det ermine the relative contribution of naturally occurring RFC activity i n 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 rela tive RFC1 expression with MTX and trimetrexate (TMTX) growth inhibitio n (Gl(50)) in a panel of cell lines used in the NCI Anticancer Drug Sc reen, Clones of transport-deficient, MTX-resistant ZR-75-1 human breas t cancer cells (MTXR ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than central cel l clones, showing that restoration of RFC activity has a significant i mpact on MTX and TMTX cytotoxicity, We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFC1 RNA levels by a quantitat ive RT-PCR assay, RFC1 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 acid ne gatively 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 additio n, compounds in the NCI Anticancer Drug Screen database with cytotoxic ity profiles which correlated with RFC1 RNA levels or with the calcula ted difference in MTX-TMTX potency were examined for MTX uptake inhibi tion and cytotoxicity in the RFC1-transfected MTXR 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-sel ected cell lines. (C) 1997 Wiley-Liss, Inc.