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• W2019995269 abstract "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 → 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. 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 → 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. methotrexate N α-(4-amino-4-deoxy-10-methylpteroyl)-N ε-(4-azido-5-[125I]iodosalicylyl)-l-lysine Chinese hamster ovary glutathioneS-transferase reduced folate carrier reverse transcriptase-polymerase chain reaction kilobase pair(s) base pair(s) peptide-specific RFC Despite the availability of newer antifolates, methotrexate (Mtx)1 continues to play an important role as an antineoplastic agent. To reach its intracellular target, dihydrofolate reductase, the preferred route of Mtx entry involves the reduced folate carrier (RFC; 1, 2). RFC transport of Mtx is critical to drug action because of its role in generating sufficient unbound intracellular antifolate to sustain maximal enzyme inhibition (1Goldman I.D. Matherly L.H. Pharmacol. Ther. 1985; 28: 77-102Crossref PubMed Scopus (205) Google Scholar). Furthermore, high levels of Mtx are also necessary for the synthesis of Mtx polyglutamates (1Goldman I.D. Matherly L.H. Pharmacol. Ther. 1985; 28: 77-102Crossref PubMed Scopus (205) Google Scholar). Defective membrane transport of Mtx by RFC has been identified as a major mechanism of Mtx resistance (1Goldman I.D. Matherly L.H. Pharmacol. Ther. 1985; 28: 77-102Crossref PubMed Scopus (205) Google Scholar, 2Sirotnak F.M. Cancer Res. 1985; 45: 3992-4000PubMed Google Scholar, 3Schuetz J.D. Matherly L.H. Westin E.H. Goldman I.D. J. Biol. Chem. 1988; 263: 9840-9847Abstract Full Text PDF PubMed Google Scholar, 4Sirotnak F.M. Moccio D.M. Kelleher L.E. Goutas L.J. Cancer Res. 1981; 41: 4442-4452Google Scholar, 5Gorlick R. Goker E. Trippett T. Steinherz P. Elisseyeff Y. Mazumdar M. Flintoff W.F. Bertino J.R. Blood. 1997; 89: 1013-1018Crossref PubMed Google Scholar, 6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 7Brigle K.E. Spinella M.J. Sierra E.E. Goldman I.D. J. Biol. Chem. 1995; 270: 22974-22979Crossref PubMed Scopus (105) Google Scholar, 8Gong M. Yess J. Connolly T. Ivy S.P. Ohnuma T. Cowan K.H. Moscow J.A. Blood. 1997; 89: 2494-2499Crossref PubMed Google Scholar, 9Roy K. Tolner B. Chiao J.H. Sirotnak F.M. J. Biol. Chem. 1998; 273: 2526-2531Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 7873-7879Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Zhao R. Assaraf Y. Goldman I.D. J. Biol. Chem. 1998; 273: 19065-19071Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Transport alterations can manifest as reduced rates of carrier translocation (reducedV max), decreased affinities for transport substrates (increased K t), or both, and may involve decreased levels of normal RFC (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar) or the expression of structurally altered RFC proteins (7Brigle K.E. Spinella M.J. Sierra E.E. Goldman I.D. J. Biol. Chem. 1995; 270: 22974-22979Crossref PubMed Scopus (105) Google Scholar, 8Gong M. Yess J. Connolly T. Ivy S.P. Ohnuma T. Cowan K.H. Moscow J.A. Blood. 1997; 89: 2494-2499Crossref PubMed Google Scholar, 9Roy K. Tolner B. Chiao J.H. Sirotnak F.M. J. Biol. Chem. 1998; 273: 2526-2531Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 7873-7879Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Zhao R. Assaraf Y. Goldman I.D. J. Biol. Chem. 1998; 273: 19065-19071Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). For instance, in Mtx-resistant K562 (K500E) cells, impaired Mtx transport is accompanied by decreased RFC transcripts and protein (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar). A G to A transition at position 890 of the murine RFC cDNA resulted in a substitution of serine 297 by asparagine and a selective decrease in Mtx binding affinity (∼4-fold) without effects on other antifolate analogs (aminopterin, 10-ethyl-10-deazaaminopterin; Ref. 9Roy K. Tolner B. Chiao J.H. Sirotnak F.M. J. Biol. Chem. 1998; 273: 2526-2531Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Likewise, replacement of serine 46 by asparagine (10Zhao R. Assaraf Y.G. Goldman I.D. J. Biol. Chem. 1998; 273: 7873-7879Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) or glutamate 45 by lysine (11Zhao R. Assaraf Y. Goldman I.D. J. Biol. Chem. 1998; 273: 19065-19071Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) in murine RFC resulted in greater impairment of uptake for Mtx than (6S)-5-formyl tetrahydrofolate. In severely transport defective L1210 cells (MtxrA), loss of transport activity appeared to reflect a single (G to C) point mutation at nucleotide 429 of the murine RFC cDNA sequence which resulted in the substitution of proline 130 by alanine (7Brigle K.E. Spinella M.J. Sierra E.E. Goldman I.D. J. Biol. Chem. 1995; 270: 22974-22979Crossref PubMed Scopus (105) Google Scholar). However, these cells also contained a wild-type RFC allele that was not transcribed. A silent wild-type RFC allele was described for Mtx-resistant MOLT-3 cells (MOLT-3/Mtx10,000; Ref. 8Gong M. Yess J. Connolly T. Ivy S.P. Ohnuma T. Cowan K.H. Moscow J.A. Blood. 1997; 89: 2494-2499Crossref PubMed Google Scholar). Moreover, two mutations in the RFC coding region were detected which resulted in the creation of new stop codons and synthesis of truncated nonfunctional RFCs (8Gong M. Yess J. Connolly T. Ivy S.P. Ohnuma T. Cowan K.H. Moscow J.A. Blood. 1997; 89: 2494-2499Crossref PubMed Google Scholar). In this report, the molecular mechanisms responsible for the transport-impaired phenotype (∼3% of wild-type) of Mtx-resistant (∼243-fold) CCRF-CEM (CEM/Mtx-1;12) cells were examined. We show that although the levels of RFC transcripts are essentially unchanged from wild-type cells, there is a complete loss of RFC protein due to early translation termination and increased turnover of a double mutant RFC protein. The residual transport activity previously described in this transport-impaired line (12Matherly L.H. Angeles S.M. McGuire J.J. Biochem. Pharmacol. 1993; 46: 2185-2195Crossref PubMed Scopus (26) Google Scholar) presumably reflects extremely low levels of wild-type RFC and/or, possibly, non-RFC modes of Mtx uptake (13Sirotnak F.M. Goutas L.J. Jacobsen D.M. Mines L.S. Barrueco J.R. Gaumont Y. Kisliuk R.L Biochem. Pharmacol. 1987; 36: 1659-1667Crossref PubMed Scopus (36) Google Scholar, 14Henderson G.B. Strauss B.P. Cancer Res. 1990; 50: 1709-1714PubMed Google Scholar, 15Sierra E.E. Brigle K.E. Spinella M.J. Goldman I.D. Biochem. Pharmacol. 1997; 53: 223-231Crossref PubMed Scopus (56) Google Scholar). [α-32P]dCTP (3000 Ci/mmol) and [α-35S-thiol]dATP (1400 Ci/mmol) were obtained from NEN Life Science Products Inc. [3′,5′,7-3H]Mtx (20 Ci/mmol) and [4,5-3H]leucine (120 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA). Unlabeled Mtx was provided by the Drug Development Branch, NCI, National Institutes of Health, Bethesda, MD. Both labeled and unlabeled Mtx were purified by high performance liquid chromatography prior to use (16Fry D.W. Yalowich J.C. Goldman I.D. J. Biol. Chem. 1982; 257: 1890-1896Abstract Full Text PDF PubMed Google Scholar). GW1843U89 (17Duch D.S. Banks S. Dev I.K. Dickerson S.H. Ferone R. Heath L.S. Humphreys J. Knick V. Pendergest W. Singer S. Smith G.K. Waters K. Wilson H.R. Cancer Res. 1993; 53: 810-818PubMed Google Scholar) was obtained from Glaxo-Wellcome Pharmaceuticals (Research Triangle Park, NC). Sequenase version 2.0 and reagents for dideoxynucleotide sequencing were from U. S. Biochemical Corp. (Cleveland, OH). Restriction and modifying enzymes were obtained from Promega (Madison, WI). Synthetic oligonucleotides were obtained from Genosys Biotechnologies, Inc. (The Woodlands, TX). Wild-type CCRF/CEM and transport-impaired CEM/Mtx (18Rosowsky A. Lazarus J. Yuan G.C. Beltz W.R. Mangini L. Abelson H.T. Modest E.J. Frei E. Biochem. Pharmacol. 1980; 29: 648-652Crossref PubMed Scopus (115) Google Scholar) lymphoblastic leukemia lines were gifts of Dr. Andre Rosowsky (Boston, MA). Cells were cloned in soft agar (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 19Matherly L.H. Czajkowski C.A. Angeles S.M. Cancer Res. 1991; 51: 3420-3426PubMed Google Scholar) and clonal lines (designated CEM-4 and CEM/Mtx-1 for the parental and Mtx-resistant cells, respectively) were used for all experiments. Transport-deficient K500E cells were selected from wild-type K562 cells by cloning in soft agar with 500 nm Mtx (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar). K500E cells were transfected with wild-type RFC (KS43) to generate the K43-1 and K43-6 sublines, as described previously (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar). The cell lines were maintained in RPMI 1640 medium as described previously (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 12Matherly L.H. Angeles S.M. McGuire J.J. Biochem. Pharmacol. 1993; 46: 2185-2195Crossref PubMed Scopus (26) Google Scholar). Transport-defective Mtx-resistant Chinese hamster ovary (CHO) cells, MtxRIIOuaR2-4 (20Flintoff W.F. Nagainis C.R. Arch. Biochem. Biophys. 1983; 223: 433-440Crossref PubMed Scopus (37) Google Scholar), were a gift of Dr. Wayne Flintoff (London, Ontario, Canada). Cells were grown in α-minimal essential medium with 10% iron-supplemented bovine calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). The pC43/10 CHO line was derived from MtxRIIOuaR2-4 cells by transfection with the full-length human RFC cDNA (KS43; Ref. 21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). CHO cells were grown as monolayers for transfection and general maintenance; for transport experiments, cells were grown in suspension in spinner flasks. Genomic DNAs were isolated from cultured cells using the PuregeneTM DNA isolation kit from Gentra Biosystem, Inc. (Minneapolis, MN). Aliquots (10 μg) were digested with restriction enzymes (either BamHI orHindIII), fractionated on a 0.6% agarose gel, and blotted onto a nylon membrane (Genescreen Plus, DuPont), following standard protocols (22Maniatis T. Fritsh E. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Total RNA was isolated from log phase cells using the TRIzol Reagent (Life Technologies, Inc.). RNA samples were analyzed on a formaldehyde-agarose gel, exactly as described previously (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Equal loading was established by probing with 32P-labeled β-actin cDNA or by staining with ethidium bromide. All membranes were hybridized with 32P-labeled full-length RFC cDNA and processed as described previously (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). RFC cDNAs from parental and CEM/Mtx-1 cells were synthesized from total RNA with random hexamers using a RT-PCR kit from Perkin-Elmer. Four sets of PCR primers were used to generate overlapping partial cDNAs spanning the entire RFC coding region. The PCR primers for RFC cDNA amplification are shown in Table I. PCR conditions were 94 °C for 30 s, 63 °C for 45 s, and 72 °C for 1 min (35 cycles), and ending with 72 °C for 7 min (1 cycle). PCR products were subcloned into the pCR 2.1 plasmid using the T-A cloning kit (Invitrogen) and the nucleotide sequences were determined by dideoxynucleotide sequencing (23Sanger F. Niklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52503) Google Scholar). RT-PCR reactions were repeated 2–3 times for regions containing the P1 and P2mutations and for each primer set, multiple cDNA clones were sequenced.Table IPCR primers for RFC cDNA amplificationPrimerSequencePositionP15′-tcgcctattcctcctacatct401 → 421P35′ -gtatgggtcgctctgtctctg1572 → 1552P45′ -gatacggccaggggagagctt120 → 140P75′ -gccagcgagatgtagttgagcgt572 → 550P85′ -cagtgtcaccttcgtcccctccg−46 → −24P95′ -cggaggagaaggcagcacggca1427 → 1449P105′ -cacccacctcttccagcaacaaa2024 → 2002Primer positions are relative to the ATG translation start. Open table in a new tab Primer positions are relative to the ATG translation start. Genomic fragments containing point mutations identified in the CEM/Mtx-1 cDNAs were PCR amplified and the PCR products subcloned and sequenced as described above. Primers for genomic amplification were based on the human RFC cDNA (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and gene (24Tolner B. Roy K. Sirotnak F.M. Gene (Amst.). 1998; 211: 331-341Crossref PubMed Scopus (53) Google Scholar, 25Zhang L. Wong S.C. Matherly L.H. Biochim. Biophys. Acta. 1998; 1442: 389-393Crossref PubMed Scopus (22) Google Scholar) nucleotide sequences. For amplifying the fragment containing the P1mutation, a nested PCR approach was used. In the primary PCR reaction, two RFC intron-specific primers, RFC-IP1 (5′-ctgcagaccatcttccaaggtgccctga; upstream of the splice acceptor site at −49) and RFC-IP2 (5′-gcagaccatcttccaaggtgccctga; downstream of the splice donor site at 189), were used. For the secondary nested PCR reaction, the primers used were the exon-specific primer P8 (Table I) and another intron-specific primer RFC-IP3 (5′-acctactggtgctgctgcccctgc; downstream of the splice donor site at 189). The fragment containing the P2 mutation was amplified with intron-specific RFC-IP4 (5′-gcggcagcattgctaacacctggtg; upstream of the splice acceptor site at 190) and exon-specific P7 (Table I) primers. PCR conditions for amplifying genomic DNAs were 94 °C for 10 s, 63 °C for 60 s, and 72 °C for 60 s (35 cycles), and 1 cycle of 72 °C for 7 min. Constructs containing the P1 (RFCP1) or P2(RFCP2) mutations were prepared by restriction digesting CEM/Mtx-1 RFC mutant cDNAs with Eco72/ApaI (for P1) or NcoI/ApaI (for P2). The fragments were subcloned intoEco72/ApaI- orNcoI/ApaI-digested wild-type (KS43) RFC in pBluescript SK(−) (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The mutated RFC cDNAs were excised withBamHI and XhoI, and the ∼2-kb fragments subcloned into a BamHI/XhoI-digested pCDNA3 expression vector. The construct containing bothP1 and P2 mutations (RFCP1+P2) was prepared by ligating the Eco72/ApaI RFCP1 fragment intoEco72/ApaI-digested RFCP2 in pCDNA3. Mutant constructs were transfected into transport defective MtxRIIOuaR2-4 and K500E cells as described previously (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar,21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). G418-resistant clones were expanded and screened for RFC transcripts (Northern), immunoreactive RFC protein (Western), and [3H]Mtx uptake. The complete coding sequence of the KS43 RFC cDNA (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) was subcloned into a pGEX glutathione S-transferase (GST) fusion vector (Pharmacia Biotech, Piscataway, NJ). Following transformation of Escherichia coli (BL21) cells and induction by isopropyl-β-d-thiogalactoside (0.5 mm) for 4 h at room temperature, GST-RFC fusion proteins were purified from bacterial lysates by affinity chromatography using glutathione-Sepharose 4B (Pharmacia Biotech), as recommended by the manufacturer. Authenticity and purity of the purified RFC fusion protein were confirmed by Coomassie Blue staining and Western analysis with anti-GST (Pharmacia Biotech) and RFC peptide-specific (RFC/ps; Ref. 26Wong S.C. Zhang L. Proefke S.A. Hukku B. Matherly L.H. Biochem. Pharmacol. 1998; 55: 1135-1138Crossref PubMed Scopus (17) Google Scholar) antibodies. Anti-GST-RFC antiserum was raised in rabbits using purified GST-RFC fusion protein as antigen (Pocono Rabbit Farms and Laboratories, Canadensis, PA). Both immune and preimmune sera were purified on protein A-agarose columns prior to use (27Harlowe E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). Plasma membranes were prepared by differential centrifugation (19Matherly L.H. Czajkowski C.A. Angeles S.M. Cancer Res. 1991; 51: 3420-3426PubMed Google Scholar, 28Matherly L.H. Angeles S.M. Czajkowski C.A. J. Biol. Chem. 1992; 267: 23253-23260Abstract Full Text PDF PubMed Google Scholar). Where noted, particulate membrane fractions were additionally purified on discontinuous sucrose gradients (19Matherly L.H. Czajkowski C.A. Angeles S.M. Cancer Res. 1991; 51: 3420-3426PubMed Google Scholar, 28Matherly L.H. Angeles S.M. Czajkowski C.A. J. Biol. Chem. 1992; 267: 23253-23260Abstract Full Text PDF PubMed Google Scholar). Plasma membrane purity and endoplasmic reticulum contamination of crude particulate and sucrose density gradient-purified membranes were established by 5′-nucleotidase (29Bodansky T. Schwartz M.K. J. Biol. Chem. 1963; 238: 3420-3427Abstract Full Text PDF PubMed Google Scholar) and NADPH-cytochrome c reductase (30Green D.E. Mii S. Kohout P.M. J. Biol. Chem. 1955; 217: 551-567Abstract Full Text PDF PubMed Google Scholar) assays, respectively. Membrane proteins were electrophoresed on 7.5% gels in the presence of SDS (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar) and electroblotted onto polyvinylidene difluoride membranes (DuPont) for detection with protein A-purified GST-RFC antibody and enhanced chemiluminescence (Pierce, Rockford, IL). A few experiments employed RFC peptide-specific (RFC/ps) antibody (26Wong S.C. Zhang L. Proefke S.A. Hukku B. Matherly L.H. Biochem. Pharmacol. 1998; 55: 1135-1138Crossref PubMed Scopus (17) Google Scholar). Light emission was recorded on x-ray film with various exposure times, and the signal was analyzed with a computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). For some experiments, the heterogeneously glycosylated RFCs were enzymatically deglycosylated with N-glycosidase F (Boehringer Mannheim), as described previously (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 28Matherly L.H. Angeles S.M. Czajkowski C.A. J. Biol. Chem. 1992; 267: 23253-23260Abstract Full Text PDF PubMed Google Scholar). Cell surface RFC proteins in wild-type CCRF-CEM and CEM/Mtx-1 cells (1 × 108 cells/labeling condition) were photoaffinity labeled usingN α-(4-amino-4-deoxy-10-methylpteroyl)-N ε-(4-azido-5-[125I]iodosalicylyl)-l-lysine (APA-[125I]ASA-Lys), as described previously (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar,21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Specificity of labeling was established by performing identical incubations in the presence of 100 μm aminopterin. Equal aliquots of labeled proteins were electrophoresed on a 4–10% gradient gel in the presence of SDS (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar). The gel was dried and exposed to x-ray film. Initial [3H]Mtx uptake rates were determined over 180 s using 1–2 × 107 cells/ml (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 12Matherly L.H. Angeles S.M. McGuire J.J. Biochem. Pharmacol. 1993; 46: 2185-2195Crossref PubMed Scopus (26) Google Scholar, 19Matherly L.H. Czajkowski C.A. Angeles S.M. Cancer Res. 1991; 51: 3420-3426PubMed Google Scholar,21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and a Mtx concentration of 0.5 μm. The levels of intracellular radioactivity were expressed as picomoles/mg of protein, calculated from direct measurements of radioactivity and protein contents of the cell homogenates. Protein assays were by the method of Lowry et al. (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Kinetic constants (K tand V max) were calculated from Lineweaver-Burk plots. Impaired Mtx Transport in CEM/Mtx-1 Cells Is Independent of Changes in RFC Transcripts or Gene Structure—Northern analysis of total RNAs from parental CCRF-CEM (CEM-4) and Mtx-resistant CEM/Mtx-1 cells showed that essentially identical levels of a major 3.1-kb RFC mRNA transcript were expressed (Fig.1) despite a ∼33-fold difference in relative Mtx transport (12Matherly L.H. Angeles S.M. McGuire J.J. Biochem. Pharmacol. 1993; 46: 2185-2195Crossref PubMed Scopus (26) Google Scholar). Although a 1-kb RNA species hybridized with the RFC cDNA in parental cells and a unique 9.5-kb band was detected in CEM/Mtx-1 cells (Fig. 1), the significance of these forms is not clear. These bands were still present even when poly(A) mRNAs were used for Northern analysis (data not shown). Restriction analysis (BamHI or HindIII) of genomic DNAs from CEM-4 and CEM/Mtx-1 cells did not reveal any major alterations in RFC gene organization or copy number between the lines (data not shown). Expression of RFC protein in plasma membranes from parental and CEM/Mtx-1 cells was analyzed by Western blotting using antibody to recombinant RFC fusion protein (GST-RFC) and chemiluminescence detection, and by photoaffinity labeling with APA-[125I]ASA-Lys (6Wong S.C. McQuade R. Proefke S.A. Bhushan A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). For both methods, a broadly migrating RFC band centered at ∼85 kDa was identified in parental cells (Fig. 2,left panel, and Fig. 3, respectively). Identical results were obtained on Western blots with peptide-specific (RFC/ps) antiserum (not shown). Slight differences were seen in the relative migrations for RFC, reflecting the different gel systems used for separation (7.5% for the Westernversus 4–10% for the photoprobe experiments). By both approaches, the major bands identified as RFC were converted to a single ∼65-kDa deglycosylated form by treatment withN-glycosidase F (shown for the immunoblotted RFC in parental CCRF-CEM cells; Fig. 2, right panel). This is the size predicted from the RFC cDNA sequence (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 33Moscow J.A. Gong M. He R. Sgagias M.K. Dixon K.H. Anzick S.L. Mettzer P.S. Cowan K.H. Cancer Res. 1995; 55: 3790-3794PubMed Google Scholar, 34Murray R.C. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 1996; 271: 19174-19179Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). By contrast, in CEM/Mtx-1 cells none of the ∼85-kDa RFC protein was detected either by Western blotting with anti-GST-RFC (Fig. 2) or peptide-specific antiserum (not shown), or by photoaffinity labeling with APA-[125I]ASA-Lys (Fig. 3). However, an unidentified 42-kDa protein was specifically labeled with the photoprobe (Fig. 3). Although there were no changes in the background staining on Western blots following treatment of CEM/Mtx-1 proteins withN-glycosidase F, the 42-kDa photolabeled band was converted to ∼37 kDa by this treatment (not shown).Figure 3Photoaffinity labeling of RFC. Equal numbers (1 × 108) of parental CCRF-CEM (CEM-4) and CEM/Mtx-1 cells were labeled with APA-[125I]ASA-Lys in the presence (+) or absence (−) of 100 μm aminopterin (AMT) at 0 °C. Labeled proteins were extracted in 1% Triton X-100 and equal aliquots were analyzed on 4–10% gradient gel. The gel was dried and exposed to x-ray film. Molecular masses of protein standards are indicated.View Large Image Figure ViewerDownload (PPT) The RFC coding sequences from parental CCRF-CEM and CEM/Mtx-1 cells were examined by RT-PCR and dideoxynucleotide sequencing of the PCR products. Four primer sets were used to amplify the entire RFC coding sequence (P1/P3, P4/P7, P8/P7, and P9/P10; TableI). Three alterations were identified in a 572-bp segment (positions −23 to 549, where 1 is the translational start site) amplified from CEM/Mtx-1 cells by primer set P7/P8 and encoding the RFC amino terminus. These include two G to A point mutations at positions 130 (designated P1; nucleotide position 1 is the translation start) and 380 (P2) in all of the 16 CEM/Mtx-1 clones sequenced, and a 4-bp (CATG) insertion at position 191 in 3 of the clones. By contrast, none of the 9 cDNA clones amplified with P7/P8 from parental CCRF-CEM cells contained any alterations from wild-type RFC sequence (21Wong S.C. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1995; 270: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 33Moscow J.A. Gong M. He R. Sgagias M.K. Dixon K.H. Anzick S.L. Mettzer P.S. Cowan K.H. Cancer Res. 1995; 55: 3790-3794PubMed Google Scholar, 34Murray R.C. Williams F.M.R. Flintoff W.F. J. Biol. Chem. 1996; 271: 19174-19179Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Analogous results were obtained by amplification of a fragment containing the P2 locus (positions 141–549) with the P4/P7 primer set (21/23 with a P2 mutation, including 7 with insertion at position 191). However, 2 of 23 clones derived from CEM/Mtx-1 also contained wild-type sequence at this position. All of the 15 clones amplified from par" @default.
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• W2019995269 title "Impaired Membrane Transport in Methotrexate-resistant CCRF-CEM Cells Involves Early Translation Termination and Increased Turnover of a Mutant Reduced Folate Carrier" @default.
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