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Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier. 1999

S C Wong, and L Zhang, and T L Witt, and S A Proefke, and A Bhushan, and L H Matherly
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.

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.

UI MeSH Term Description Entries
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D008727 Methotrexate An antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of TETRAHYDROFOLATE DEHYDROGENASE and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA. Amethopterin,Methotrexate Hydrate,Methotrexate Sodium,Methotrexate, (D)-Isomer,Methotrexate, (DL)-Isomer,Methotrexate, Dicesium Salt,Methotrexate, Disodium Salt,Methotrexate, Sodium Salt,Mexate,Dicesium Salt Methotrexate,Hydrate, Methotrexate,Sodium, Methotrexate
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010641 Phenotype The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment. Phenotypes
D002352 Carrier Proteins Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes. Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D002462 Cell Membrane The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells. Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D005492 Folic Acid A member of the vitamin B family that stimulates the hematopoietic system. It is present in the liver and kidney and is found in mushrooms, spinach, yeast, green leaves, and grasses (POACEAE). Folic acid is used in the treatment and prevention of folate deficiencies and megaloblastic anemia. Pteroylglutamic Acid,Vitamin M,Folacin,Folate,Folic Acid, (D)-Isomer,Folic Acid, (DL)-Isomer,Folic Acid, Calcium Salt (1:1),Folic Acid, Monopotassium Salt,Folic Acid, Monosodium Salt,Folic Acid, Potassium Salt,Folic Acid, Sodium Salt,Folvite,Vitamin B9,B9, Vitamin
D006224 Cricetinae A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS. Cricetus,Hamsters,Hamster
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000964 Antimetabolites, Antineoplastic Antimetabolites that are useful in cancer chemotherapy. Antineoplastic Antimetabolites

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