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

Abstract

A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate fMTXI).We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels ofhFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of I3Hlmethotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity. (J. Clin. Invest. 1993.91:1289-1294

Resistance to antifolates

“…When FR expression is increased in cells with functioning RFC, the impact on antifolate activity and folate growth requirement depends on the relative expression of the transporters and the relative affinities of the folate for the two processes (Westerhof et al, 1991). With sufficient expression of FR, even drugs such as MTX, with low affinity for these proteins relative to folic acid, can be delivered into cells at rates comparable to those mediated by RFC (Dixon et al, 1992;Spinella et al, 1995); and growth inhibition can be enhanced (Chung et al, 1993). The human KB nasopharyngeal epidermoid line with high-level expression of FR-a is highly sensitive to MTX, and transport is mediated largely by this process (Deutsch et al, 1989).…”

“…The impact of FR expression on the activity of antifolates is influenced by the folate present and its concentration in vitro or in vivo and the relative affinities of the folate and antifolate for this transporter. Hence, in standard media with folic acid levels of 2.3 mm, transport of MTX via FRs is markedly suppressed, with a comparable decrease in growth inhibition by this agent due to the 300-fold higher affinity of folic acid for the receptor (Kane et al, 1986;Chung et al, 1993). Likewise, activity of the nonpolyglutamatable TS inhibitor, CB300638 which has a very high affinity for folate receptor but a low affinity for RFC, was marked suppressed when 1 mm folic acid was added to medium containing either 1 or 20 nm 5-CHO-THF (Theti et al, 2003).…”

Sorting and Function of the Human Folate Receptor Is Independent of the Caveolin Expression in Fisher Rat Thyroid Epithelial Cells

“…Cell surface folic acid binding assay Cell surface binding assays, using [ 125 I] labeled folic acid, were carried out as described (Chung et al, 1993), using 10 5 cpm ligand in 1 ml DMEM plus 50 µg/ml BSA in a 35 mm well plate. The cells (10 6 ) were plated 18 hours prior to the experiment.…”

“…This resulted in a final folate concentration of approximately 1-10 nM (Chung et al, 1993). Photographs of the cells were taken after 14 days.…”

“…The hFR is an essential component of one of two different transport systems that have been described for folate and antifolate transport (Elwood et al, 1986;Antony, 1992;Chung et al, 1993). The human KB cell hFR is the bestcharacterized isoform of folate receptors (Elwood et al, 1986); it is the subject of this paper.…”