CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 M to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type K m value for MTX transport but a 31-fold and 9-fold lower K m values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G 3 A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 M folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7-and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a ϳ100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steadystate level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K m for folic acid; (b) 6-fold increased transport K m for GW1843; and (c) a slightly increased transport V max for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 3 Leu, Glu-45 3 Lys, and Ser-46 3 Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.Reduced folates are essential cofactors that function as onecarbon donors necessary for the biosynthesis of purines, thymidine, and glycine (1). Unlike prokaryotes, animal cells are devoid of de novo biosynthesis of folic acid and therefore meet their folate requirements by folate uptake from exogenous sources (1). Several transport systems have been described in various mammalian model cell lines that can accommodate transport of folates and their folate-based chemotherapeutic agents including methotrexate (MTX) 1 (2-4). (a) The reduced folate carrier (RFC) is the major uptake route that functions as a bidirectional anion exchanger (5, 6) with a high affinity (K m ϭ 0.3-5 M) for reduced folates and MTX but low affinity (K m ϭ 200 -400 M) for folic acid (4, 5, 7). (b) Folate receptors, glycosylphosphatidylinositol membrane-anchored proteins that mediate the unidirectional uptake of folates, display a high affinity for folic acid and 5-methyltetrahydrofolate (K D ϭ 1-10 nM) but lower affinity (K D ϭ 10 -300 nM) for other reduced folates and MTX (8 -11). (c) An apparently independent transport system with optimal uptake activity at low pH, which recognizes folic acid, reduced folates and MTX with comparable affinities (K m ϭ 1-5 M) (12-15).The molecular cloning and the primary structures of the human, mouse, and hamster RFC genes have been described (16 -21). Human RFC is an integral plasma membrane protein with 591 amino acids, is predicted to contain 12 transmembrane domains (TMDs), has a short N terminus ...

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Expression of TS and DPD proteins is not predictive for survival in patients with stage III colon cancer treated adjuvantly with 5-FU regimens. TS protein levels did not alter the effect of TP 53 mutation status.

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