Three mechanisms for resistance to methotrexate (Mtx) have been identified in Chinese hamster ovary (CHO) cells selected from resistance to this drug. First-step selections produce cells with either an apparent structural alteration in the enzyme dihydrofolate reductase (class I), or a decreased permeability to the drug (class II). Mutagenesis with ethyl methanesulfonate increases the proportion of Mtx-resistant cells 5-10-fold. Second-step selections to higher resistance using class I resistant cells as parents results in cells with an increased activity of the reductase enzyme (class III) with no apparent further qualitative alterations in the enzyme. All three classes of resistant cells retain their Mtx-resistant phenotype when cultured under nonselectivve conditions.

Chinese hamster ovary and rat myoblast cells resistant to the toxic action of methylglyoxal bis guanylhydrazone (MGBG), an antimitotic agent and inhibitor of polyamine synthesis, have been isolated by single step selection. Mutagenesis with ethyl methanesulfonate increases the recovery of the variants at least 30-fold. Intracellular accumulation of MGBG is greatly reduced in resistant cells. This property is accompanied by a 99% decrease in the uptake of all three naturally occurring polyamines. Both the resistant phenotype and the defect in polyamine transport behave recessively in somatic cell hybrids.

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The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.Mammalian cells require reduced folates for several biochemical pathways involving purine, pyrimidine, and amino acid metabolism. Since mammals cannot synthesize folates, these compounds must be obtained in the diet and imported by cells via either the glycosylphosphatidylinositol-linked folate receptor (1-5) or the reduced folate carrier (RFC), 1 an integral membrane protein (6 -9). A third, low pH-dependent component for folate transport has been reported (10, 11), but its involvement in folate delivery is not clearly understood. The RFC is the main route for transport of 5-methyltetrahydrofolate, the major circulating folate in the bloodstream, 5-formyltetrahydrofolate (folinic acid), and the folate analog, methotrexate (Mtx) (9,(12)(13)(14).The rfc gene has been cloned from hamster (15), human (16 -19), murine (20), and rat 2 sources. The identity of the rfc gene was verified by cDNA transfections that restored Mtx sensitivity to RFC-deficient cells (15)(16)(17)(18)(19)(20). The human RFC protein is 591 amino acids as deduced from its cDNA sequence, and the hydropathy profile predicts 12 transmembrane-spanning (TM) domains with the amino and carboxyl termini located in the cytoplasm. Recently, the cytoplasmic location of the carboxyl-terminal tail has been confirmed for the human RFC (22). The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails than the human RFC (15-20), although all three RFCs share about 68% identical or similar amino acid residues. The human RFC is glycosylated at an asparagine residue in the first extracellular loop (9,22,23), and while the hamster RFC contains the conserv...

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