Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier.
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 approximately 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 --> arginine) and 380 (P2; changes serine 127 --> 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.
Wong
S C
SC
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
Zhang
L
L
Witt
T L
TL
Proefke
S A
SA
Bhushan
A
A
Matherly
L H
LH
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D019008:Drug Resistance, Neoplasm; D005492:Folic Acid / Q000378:metabolism*; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D010641:Phenotype; D014176:Protein Biosynthesis; D058978:Reduced Folate Carrier Protein; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; D005492:Folic Acid; D008727:Methotrexate