Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

Abstract

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.The folic acid analog methotrexate (Mtx) is a cytotoxic agent to dividing mammalian cells which exerts its effect by inhibiting the enzyme dihydrofolate reductase (2). To achieve this function, the drug must be transported across the cell membrane and released intracellularly. It is believed that this transport process is mediated by an energy-dependent carrier system requiring anion exchange (14). There is conflicting evidence from various studies as to whether the analog is transported by the same system that allows folic acid to enter the cell or whether a separate system for reduced folate is employed. Kinetic and competition analyses in L1210 mouse cells have shown that two systems exist under one set of conditions (26, 31), while only one system exists under alternative conditions (13). In addition, some mutants are defective in uptake of both Mtx and folic acid, while others are defective in uptake of only Mtx (9, 10; T. M. Underhill and W. F. Flintoff, Somat. Cell Mol. Genet., in press). Also, some Mtx-deficient mutants have increased requirements for reduced folates to maintain growth without a concomitant increase in the requirement for folic acid (18,25,27). Taken together, these observations suggest that some but not all components may be shared in the uptake of these two compounds. In addition, some Mtx-and folatebinding proteins present in cell membranes have been identified (1,5,6,12,15,16,19,20,22), although their role in the overall uptake process is unclear. A recent report, however, indicates that a mouse membrane 45-to 48-kilodalton protein may be involved (32).In a recent report, we have described an Mtx-resistant Chinese hamster ovary (CHO) cell line, designated Mtx RII OuaR 2-4, that was unable to transport Mtx but was able to take up folic acid in a manner similar to that of wild-type cells (Underhill and Flintoff, in press). Under conditions of folic acid-free medium, this cell line required 250 times the level of folinic acid required by wild-type cells to maintain optimum growth. This requirement for high levels of folinic acid in otherwise folic acid-free medium is a common property shown by several mutants defective in Mtx uptake (17,27). Pseudorevertants selected from this cell line for growth on low levels of folinic acid retained their resistance to Mtx. Because the Mtx-resistant phenotype was a genetically recessive trait (8), it should be possible to complement thi...

Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

“…In contrast, MTX uptake by the A6 and A4 transfectants is inhibited by both cold MTX (> 90%) and folic acid (> 60%). The absence of cell surface folic acid binding and the lack of inhibition of MTX influx in the presence of excess folic acid suggest that MCF-7 cells have a hFR-independent method of accumulating MTX such as the reduced folate transporter (1)(2)(3)(4)(5)(6)(7)(8). However, transfection of MCF-7 cells with hFR appears to further increase (2.4-and 3.2-fold) their ability to internalize additional MTX.…”

“…Studies in normal and neoplastic human, and murine cells suggest that cellular resistance to methotrexate (MTX)' cytotoxicity is commonly due to a defect in MTX transport (1)(2)(3)(4)(5)(6)(7)(8). Although transport defects have been implicated in development of resistance to MTX, specific mutations or changes in expression of transport molecules have not been described.…”

Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion

“…Stable transfectants of MtxRII 5-3 cells were obtained using the Polybrene procedure described previously (44). Stable clones of the HA insertion constructs in either the 5Ј⌬RFC or 5Ј⌬RFC-EGFP background (described below) were selected and maintained in folic acid-free medium containing 10% dialyzed fetal bovine serum and 2 nM folinic acid (45). Stable clones of MtxRII 5-3 cells transfected with the 5Ј⌬RFC-EGFP fusion products were also selected with G418 (1.2 mg/ml) in ␣-medium supplemented with 10% fetal bovine serum.…”

“…This was done to determine whether the EGFP moiety affected RFC function, as it was desirable to have a tag to monitor protein expression and cellular location. MtxRII 5-3 cells were transfected with plasmid DNA from either RFC, RFC-EGFP, 5Ј⌬RFC, or 5Ј⌬RFC-EGFP constructs and selected in low folinic acid medium (45). There was no significant difference in numbers of colonies obtained for these four constructs, indicating that neither the 94 base pairs of the rfc 5Ј-UTR nor the EGFP fusion affect the ability of the transfected RFC to complement functionally the RFC-deficient cell line (Table III).…”

“…The HA-G54 stable transfectants showed Mtx sensi- tivity that was intermediate between the wild-type and the MtxRII 5-3 response (not shown). None of these transfected constructs were tested for binding or uptake of folates since previous work has shown that cells able to grow in low levels of folinic acid can bind and take up folates (15,16,(45)(46)(47).…”