A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells.

K.H Dixon,B.C Lanpher,J Chiu,K Kelley,K.H Cowan

doi:10.1016/s0021-9258(17)42301-5

K.H Dixon, B.C Lanpher + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(17)42301-5

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Abstract

Mammalian cells accumulate reduced folates and methotrexate, a folate antagonist, through the reduced-folate carrier (RFC) (Goldman, I.D., Lichtenstein, N.S., and Oliverio, V.T. (1968) J. Biol. Chem. 243, 5007-5017). This study describes the isolation and expression of a cDNA clone that restores RFC activity to human breast cancer cells defective in this transporter. The cDNA cancer cells defective in this transporter. The cDNA codes for a peptide (mRFC1) of 58 kDa, whose hydropathy plot, resembling those of mammalian sugar transporters, predicts that it may be a member of a superfamily of transporter genes. Transfection of methotrexate-resistant (MTXR) ZR-75-1 cells with an expression vector, pRFC1, that codes for this peptide restores their ability to accumulate methotrexate. Furthermore, transport of methotrexate into pRFC1-transfected cells is blocked by a 10-fold molar excess of the reduced folate, 5-formyltetrahydrofolic acid, but is unaffected by folic acid. The increase in methotrexate uptake that is observed in pRFC1-transfected MTXR ZR-75-1 cells reverses their resistance to this antitumor agent.

Highlights

Folate Carrier Activity and affinity for reduced folates and methotrexatethan it does for folic acid
From the Medical Breast Cancer Section, M e d ~ c ~ n e cancer cell line, MTXR ZR-75-1, with acquired resistance to Branch, National Cancer Institute, Nationa~ Institutes methotrexate, that is deficient in RFC activity (17-19)
The increasein methotrexate uptake thatis DHlOB electrocompetentcells (Life TechnologiesI,nc.), and thesewere observed in pRPC1-transfectedMTXR ZR-7S-1 cells re- selected for growth in 100 pg/ml ampicillin (Sigma).The complexity of verses their resistance ttohis antitumor agent

Summary

Methotrexate Sensitivity to Transport Deficient Cells*

Changes in RFC activity havebeen linked withthe acquisition of cellular resistance to methotrexate both in vivo and i n vitro (2-6, 14). Cells that lack the RFC require higher concentrations of (Received for pubIication, September 21, 1993,and inrevised reduced folate compounds fogrrowth than cells that do express form, October 22, 1993) this activity (15). From the Medical Breast Cancer Section, M e d ~ c ~ n e cancer cell line, MTXR ZR-75-1, with acquired resistance to Branch, National Cancer Institute, Nationa~ Institutes methotrexate, that is deficient in RFC activity (17-19). -tAa1rneccxdDeaNsltleesAl,earwcentasdetsdoilrsofeowos-r This study describes the isolation and expression of a their ability to accumulate methotrexate alnsdorestorestheir cDNA clone that restoreRs FC activity to human breast sensitivity tomethotrexate. Cancer cells defective in this transporter.The cDNA codes for a peptide (mRFClo)f 68 m a , whose hydropa-

MATERIALS AND METHODS

Reduced Folate Carrier cDNA

AND DISCUSSION

CLU lm YTXr