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J Biol Chem, Vol. 274, Issue 15, 10388-10394, April 9, 1999

Impaired Membrane Transport in Methotrexate-resistant CCRF-CEM Cells Involves Early Translation Termination and Increased Turnover of a Mutant Reduced Folate Carrier

So C. WongDagger §, Long ZhangDagger , Teah L. WittDagger , Susan A. ProefkeDagger , Alok Bhushan, and Larry H. MatherlyDagger §

From the Dagger  Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute and the § Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201 and the  Department of Pharmaceutical Sciences (AB), Idaho State University, Pocatello, Idaho 83209

The basis for impaired reduced folate carrier (RFC) activity in methotrexate-resistant CCRF-CEM (CEM/Mtx-1) cells was examined. Parental and CEM/Mtx-1 cells expressed identical levels of the 3.1-kilobase RFC transcript. A ~85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cells, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and sequence analysis, G to A point mutations were identified in CEM/Mtx-1 transcripts at positions 130 (P1; changes glycine 44 right-arrow arginine) and 380 (P2; changes serine 127 right-arrow asparagine). A 4-base pair (CATG) insertion detected at position 191 (in 19-30% of cDNA clones) resulted in a frameshift and early translation termination. Wild-type RFC was also detected (0-9% of clones). Wild-type RFC and double-mutated RFC (RFCP1+P2) cDNAs were transfected into transport-impaired K562 and Chinese hamster ovary cells. Although RFC transcripts paralleled wild-type protein, for the RFCP1+P2 transfectants, disproportionately low RFCP1+P2 protein was detected. This reflected an increased turnover of RFCP1+P2 over wild-type RFC. RFCP1+P2 did not restore methotrexate transport; however, uptake was partially restored by constructs with single mutations at the P1 or P2 loci. Cumulatively, our results show that loss of transport function in CEM/Mtx-1 cells results from complete loss of RFC protein due to early translation termination and increased turnover of a mutant RFC protein.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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