N10-Propargyl-5,8-dideazafolic acid (CB3717) and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI-198,583) are potent folate-based inhibitors of thymidylate synthase. We studied the membrane transport and the growth-inhibitory effects of the two thymidylate synthase inhibitors on human CCRF-CEM leukemia cells with different transport properties for folic acid, reduced folates, and methotrexate (MTX). Membrane transport of [3H]ICI-198,583 can proceed via the high affinity/low capacity reduced folate carrier as supported by findings that (a) uptake of [3H]ICI-198,583 was significantly impaired in CEM cells which have a transport defect for MTX, (b) variants of CEM cells which overproduce the reduced folate carrier system showed a concomitant increase in the uptake of [3H]ICI-198,583 as for [3H]MTX, (c) MTX inhibited transport of [3H]ICI-198,583, and (d) uptake of [3H]ICI-198,583 was inhibited after treatment of CEM cells with an N-hydroxysuccinimide ester of MTX, which is a potent inhibitor of MTX transport.

However, a membrane-associated folate-binding protein (FBP) offers another route for entry of CB3717 and ICI-198,583. CEM-FBP cells that have an elevated amount of FBP and do not have a functional reduced folate carrier were 640- and 61-fold more sensitive to CB3717 and ICI198,583, respectively, compared to control CEM cells expressing the reduced folate/MTX carrier. This high sensitivity was related to a high affinity of the FBP for CB3717 and ICI-198,583 (Kd 2–3 nm), which is only 3-fold lower than for folic acid (Kd 1 nm) but significantly higher than for MTX (Kd 100 nm). Furthermore, after incubation of CEM-FBP cells for 24 h at 10 nm [3H]ICI-198,583, the high affinity binding of the FBP for ICI-198,583 allowed a 600-fold concentrative uptake of [3H]ICI-198,583 and its conversion to polyglutamate forms.

These results indicate that multiple folate transport systems may be involved in the uptake of folate-based thymidylate synthase inhibitors.


This study was supported by the Dutch Cancer Society (Grant UUKC 89-06) and grants from the Cancer Research Campaign and the Medical Research Council to D. R. N. and A. L. J.

This content is only available via PDF.