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Blood, Vol. 89 No. 3 (February 1), 1997:
pp. 1013-1018
Defective Transport Is a Common Mechanism of Acquired Methotrexate Resistance in Acute Lymphocytic Leukemia and Is Associated With Decreased Reduced Folate Carrier Expression
By
Richard Gorlick,
Erdem Goker,
Tanya Trippett,
Peter Steinherz,
Yaroslav Elisseyeff,
Madhu Mazumdar,
Wayne F. Flintoff, and
Joseph R. Bertino
From the Program of Molecular Pharmacology and Therapeutics, and the Departments of Pediatrics, and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY; and the Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
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ABSTRACT |
Methotrexate (MTX) transport was examined in 27 patients with untreated acute lymphocytic leukemia (ALL) and 31 patients with relapsed ALL using a previously described fluorescent MTX analog (PT430) displacement assay (Blood 80:1158, 1992). Only 13% of untreated patients were considered to have impaired MTX transport, whereas more than 70% of relapsed patients had evidence of impaired MTX transport. To further characterize the basis for this defect, Northern analyses for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of nine samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. None of 15 samples with normal MTX transport had decreased RFC expression. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed to quantitate RFC mRNA expression more accurately. Decreased RFC expression was demonstrated in six of the nine samples with impaired MTX transport, confirming the results obtained by Northern blot. These data indicate decreased RFC expression associated with impaired MTX transport is observed in relapsed ALL following treatment with MTX-containing therapy.
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INTRODUCTION |
METHOTREXATE (MTX) continues to have an important role in the treatment of a variety of malignancies, including leukemia, lymphoma, choriocarcinoma, head and neck cancer, and osteogenic sarcoma.1 The usefulness of MTX in the treatment of these malignancies is limited by the development of drug resistance. In experimental tumor systems, the two common mechanisms of acquired resistance to MTX are decreased transport and amplification of the target gene for MTX, dihydrofolate reductase (DHFR).2-5 However, relatively little information is available on mechanisms of resistance to MTX in patient samples. We recently have shown that low-level gene amplification is present in approximately 30% of patients with acute lymphocytic leukemia (ALL) who relapse after treatment with this drug.6 In a previous study, blasts from two of four patients with ALL believed to be clinically resistant to MTX had evidence of impaired transport, allowing the suggestion that MTX transport impairment may also be a mechanism of acquired resistance in this disease.7
In this study, a competitive displacement flow cytometric assay using the fluorescent lysine analog of MTX, N -(4-amino - 4 -deoxy - N10 - methylpteroyl) - N - (4'flouresceinthio-carbamyl)-L-lysine(PT430) was used to measure MTX transport in blasts. This method is both sensitive and rapid, and is a useful method to detect MTX transport resistance.7 This assay has also been used by Matherly et al8 to demonstrate that decreased MTX transport may be a component of MTX resistance in childhood ALL.
In this report, we describe studies of MTX transport in the blasts of 74 leukemia patients, as measured by the PT430 competitive displacement assay. The availability of a human cDNA for the reduced folate carrier (RFC)9-12 has also allowed us to measure RFC mRNA expression by Northern blot analysis and to develop a reverse-transcriptase polymerase chain reaction (RT-PCR) assay to quantitate RFC gene expression.
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MATERIALS AND METHODS |
Preparation of patient samples.
A total of 77 blast samples (27 with newly diagnosed ALL, 31 with relapsed ALL, and 19 with AML) from 74 patients were studied. The patients ranged in age from 2 to 73 years and included pre-B-, B-, and T-ALL, as well as M2, M4, and M5 AML. All relapsed patients with ALL had been treated with MTX as part of protocols (NYII or CCG-106 for children and ALL-1 or L-20 for adults13-15 ) used at Memorial Sloan-Kettering Cancer Center (New York, NY). Two milliliters of bone marrow aspirate or 10 mL of peripheral blood in preservative-free heparin was drawn after obtaining informed consent. Mononuclear cells were isolated from blood and bone marrow by Ficoll-Hypaque density centrifugation. All samples contained more than 70% leukemic blasts. Cells were washed twice with phosphate-buffered saline. Cells (2 to 5 × 107) were pelleted and used immediately for preparation of RNA. For use in the PT430 competitive displacement assay, 3 × 107 cells were added to RPMI-1640 media supplemented with 10% fetal calf serum.
RNA isolation.
Total cellular RNA was isolated with RNAzol (BiotecX Laboratories, Houston, TX) according to the manufacturer's instructions. RNA was quantitated by determining the absorbance at 260 nm.
PT430 competitive displacement assay.
PT430 was synthesized and kindly provided by Drs Joel Wright and Andre Rosowsky of the Dana-Farber Cancer Institute using previously described methods.16,17 The PT430 assay was performed as described previously.7,8,18 Briefly, 3 × 107 cells were incubated in the presence of 2 × 10-5 mol/L PT430 for 2 hours. After washing and allowing for efflux of unbound drug (30 minutes), displacement of PT430 was measured after incubation at 37°C in the presence of the competitors MTX and trimetrexate (TMTX) both at a concentration of 5 × 10-7 mol/L for 90 minutes. Changes in fluorescence were measured by flow cytometry. PT430 displacement was corrected for the nondisplaceable component. Less than 40% displacement of PT430 by MTX was considered indicative of defective transport as was defined in the previous study.7
Northern blotting.
Thirty micrograms of total RNA was denatured in 3-(N-Morpholino)propanesulfonic acid (MOPS)-formaldehyde-formamide solution at 65°C for 15 minutes, then electrophoresed on a 1% agarose gel under denaturant conditions. After washing the gel with 10 × SSC (1 × SSC is 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate) for 30 minutes at room temperature, RNA samples were transferred to an Optitran nitrocellulose membrane (Schleicher & Schuell, Keene, NH) according to a standard protocol.19 RNA was fixed to the nitrocellulose by UV irradiation using a Stratagene cross-linker (La Jolla, CA). The DNA for the probe was prepared by digesting pHuMtxT49 with BamH1 and purifying the 2.6-kb fragment using a Geneclean Kit (Bio101, Vista, CA). The probe was labeled with [ -32P]dCTP using a random-primed DNA labeling kit (Boehringer-Mannheim, Indianapolis, IN). The blots were prehybridized for 4 hours, hybridized with probe at 42°C overnight, and washed to a final stringency of 0.1 × SSC and 0.1% sodium dodecyl sulfate (SDS) at 65°C. Autoradiography was performed by overnight exposure to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens at -70°C. The Northern blots were stripped by boiling and reprobed with 36B420 to determine loading. Radioactivity was quantitated on a Betascope 603 blot analyzer (Betagen, Waltham, MA).
Quantitative RT-PCR.
The method developed is based on a noncompetitive amplification of the target gene as compared with an internal standard ( -actin).21 Five to 10 µg of total RNA was incubated with 5 U of RNase-free DNase (Boehringer-Mannheim) for 15 minutes at 37°C, then extracted with phenol:chloroform, precipitated with 100% ethanol, washed with 75% ethanol, and resuspended in water. The RNA was denatured using methyl-mercury hydroxide and -mercaptoethanol and converted to cDNA with random hexamers using a cDNA cycle kit (Invitrogen, San Diego, CA) as per the manufacturer's instructions, including a second-strand synthesis. RNA prepared from the CCRF-CEM cell line was used as an internal standard. The cDNA was stored at -80°C before use. The cDNA was serially diluted from 1:10 to 1:106 and each dilution was used for two PCR reactions: one using RFC primers and the other using -actin primers. The oligonucleotides used as primers were synthesized by Operon Technologies, Alameda, CA. The -actin primers were BA67 and BA68, which have been described previously.21,22 The RFC primers used were as follows: RFC617, 5'-CCAAGCGCAGCCTCTTCTTCAACC; and RFC949, 5'-CCAGCAGCGTGGAGGCAGCATCTGCC; which produce an approximately 300-bp PCR product. The PCR mixture contained 10 pmol of each oligonucleotide primer, 2 nmol of each of the four deoxyoligonucleotides, 5 µCi of [-32P]dCTP, 0.125 U Taq polymerase, 2.5 µL of 10× CR buffer (Perkin-Elmer, Norwalk, CT), and 10 µL of cDNA diluted with water to a final reaction volume of 25 µL. Forty cycles of the PCR were performed at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute in a Gene-Amp PCR system 9600 (Perkin-Elmer). The PCR reaction was optimized to produce the largest linear range in the samples that had low RFC expression. Twenty microliters of each PCR product was electrophoresed on a 4% acrylamide gel in 1 × TBE buffer (1 × TBE is 89 mmol/L Tris base, 89 mmol/L boric acid, 2 mmol/L EDTA) at 120 V for 1 hour. The gel was dried for 2 hours at 80°C under vacuum (Bio-Rad Model 583 gel dryer, Richmond, CA) and radioactivity was quantitated on a Betascope 603 blot analyzer (Betagen). RFC/-actin ratios were calculated by determining the linear range for each sample, plotting the best fit line, and determining the RFC/-actin ratio at the X intercept.
Biostatistical considerations.
Equality of proportion of leukemic blasts obtained from patients with newly diagnosed ALL and from relapsed patients was tested using a binomial proportion test and two-sided P values are reported.23
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RESULTS |
The displacement of PT430 by MTX, as well as TMTX, in the 77 blast samples in addition to the blasts from 17 patients previously reported7 is displayed as histograms in Fig 1. Only 4 of 30 (13%) leukemic blast samples obtained from patients with newly diagnosed ALL had less than 40% displacement of PT430 by MTX. In contrast, blasts from 25 of 35 patients (71%) with relapsed ALL had less than 40% displacement (P < .0001). In contrast, as expected, as TMTX does not use the RFC for uptake, only 10 of 35 patients (28%) with relapsed ALL had less than 40% displacement of PT430 by TMTX (compared with 2 of 27 untreated patients). In AML, 10 of 29 (34%) samples had less than 40% displacement of PT430 by MTX. Seven of 20 patients (35%) with newly diagnosed AML and 3 of 9 (33%) with relapsed AML had less than 40% displacement. In AML, failure to displace PT430 by TMTX (10 of 29, 34%) is more common than in ALL (2 of 27, 7%) (P = .032).
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| Fig 1.
MTX transport in 94 leukemic blast samples as determined by PT430 (2 × 10-5 mol/L) displacement by MTX (5 × 10-7 mol/L) ( ) and TMTX (5 × 10-7 mol/L) ( ). (A) Newly diagnosed ALL (n = 30); (B) relapsed ALL (n = 35); (C) acute myelogenous leukemia (AML) patients (n = 29).
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| Fig 2.
Northern blot of patient samples probed with RFC and 36B4 as a loading control. (A) Patient sample (no. 26) with normal RFC expression and patient sample (no. 58) with no detectable RFC expression relative to CCRF-CEM (CEM-S) cell line. (B) Patient sample (no. 36) with decreased RFC expression relative to the CCRF-CEM (CEM-S) cell line.
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To determine the basis for the impairment of MTX transport in blasts from ALL patients in relapse, considered to be clinically resistant to this drug, we measured expression of the RFC. There was sufficient material available to perform Northern analysis on RNA obtained from blasts of 9 patients with impaired transport as measured by PT430 displacement and in 15 patients who did not show impaired transport of MTX. Representative Northern blots are shown in Fig 2, which illustrates patient samples with normal, decreased, and no detectable RFC expression relative to a human lymphoblast CCRF-CEM cell line, which is sensitive to MTX (EC50 = 15 nmol/L for 120-hour exposure).24 Of 9 transport-defective samples, 5 had decreased (30% to 90%) and 1 had no detectable RFC expression compared with the RNA from the CCRF-CEM cell line. The remaining 3 transport-defective samples tested had no decrease in RFC expression. None of the 15 samples with normal MTX transport had decreased RFC mRNA expression.
As the results of Northern analysis and quantitation of radioactivity using the Betascope were semiquantitative, a more sensitive method for quantitating RFC expression using RT-PCR was developed and standardized. This assay was performed on the 24 samples in which RFC expression had been measured by Northern analysis. A representative assay is shown in Fig 3 and the results are summarized in Fig 4. Six of nine transport-defective samples that had evidence of decreased RFC expression by Northern blot all had greater than a threefold reduction in RFC/actin ratios compared with a CCRF-CEM cell line. The remaining three transport-defective samples, as well as the 15 samples with normal MTX transport, had less than twofold reduction in the RFC/actin ratios as compared with the CCRF-CEM cell line. A wide range of RFC/actin ratios was observed in the transport normal samples (0.1 to 1.8).
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| Fig 3.
Quantitative RT-PCR of patient samples for RFC expression. Serially diluted cDNA is amplified by PCR with primers for the RFC and -actin, which is used as a standard. Patient samples with normal RFC expression (no. 26), no detectable RFC expression (no. 58), and decreased RFC expression (no. 36) are displayed with the CCRF-CEM (CEM-S) cell line.
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| Fig 4.
The ratio of RFC/actin expression as determined by quantitative RT-PCR in transport-defective (PT430 displacement by MTX < 40%) (n = 9) and transport-normal samples (PT430 displacement by MTX < 40%) (n = 15). Black bar indicates the median of each group.
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DISCUSSION |
Folates may be transported into cells by two systems: the folate receptors and the RFC. The folate receptors have a higher affinity for folic acid as compared with the reduced folates, while the RFC has a higher affinity for reduced folates and MTX as compared with folic acid.25 The RFC is the predominant transporter of MTX in most malignant cell types.26
The cDNA for the RFC was recently cloned from hamster cells by identifying a cDNA clone that complemented a MTX transport-defective Chinese hamster ovary cell line's ability to grow in media with low amounts of folinic acid (5-formyl tetrahydrofolate) as the sole folate source.27 A homologous cDNA was also identified from murine cDNA by identifying a clone that complemented a MTX transport-defective breast cancer cell line's ability to grow in the presence of folinic acid and TMTX.28 The murine and hamster cDNA sequences were subsequently used by investigators to identify the human homolog of the RFC.9-12
In this study, defective MTX transport (PT430 displacement by MTX < 40%) was found in 71% of blasts from patients with ALL who relapsed after treatments that contained MTX, while 13% of blast samples had evidence of low MTX transport at diagnosis. Evidence of decreased TMTX transport was found in 28% of relapsed ALL patients. As TMTX has been reported to share in the multidrug resistance (MDR) phenotype, it is possible that blasts from these patients were also expressing p-glycoprotein, the product of the MDR-1 gene, as all had received therapy that could result in increased MDR expression.29,30 Another theoretical possibility is a mutant DHFR that binds normally to MTX and PT430, but has decreased affinity for TMTX. This possibility is unlikely, as DHFR cDNA from blasts from relapsed ALL patients have been sequenced without evidence of mutations.31
A quantitative RT-PCR method based on the assay described by Horikoshi et al21 was developed to measure RFC expression. In six of nine transport-defective samples, decreased expression of the RFC gene was observed both by Northern analysis and quantitative RT-PCR. The association of RFC expression and MTX sensitivity, presumably secondary to alterations in transport, has been reported previously in cell lines.32 Three of nine transport-defective samples were found to have normal levels of RFC expression by Northern blot, as well as quantitative RT-PCR. Sequencing of the entire cDNA for the RFC in these three samples is currently in progress to determine if there are mutations in the coding sequence that might explain the low level of MTX transport in these samples. A mutation in the RFC of a murine L1210 leukemia cell line resulted in decreased MTX uptake and MTX resistance despite an approximately fivefold increase in RFC message.33 Defective MTX transport associated with decreased RFC expression is therefore common in relapsed ALL following treatment with MTX-containing therapy.
In AML, previous studies have attributed intrinsic resistance to MTX to defects in polyglutamylation of this drug.34,35 This current study provides evidence that defective MTX transport may also contribute to intrinsic resistance in AML, as impaired MTX transport was found in 35% of blasts from AML patients at the time of diagnosis. In addition, evidence of defective TMTX transport is also observed in 25% of AML patients at diagnosis. This also may be secondary to expression of p-glycoprotein, which has been reported to be capable of effluxing TMTX and has a high incidence of expression in untreated patients with this disease.36,37 We are currently testing the correlation of impaired displacement of PT430 by TMTX also with decreased daunorubicin uptake.
The high incidence of MTX transport resistance found in relapsed ALL patients allows the development of novel strategies to overcome this type of resistance. One such strategy that we are exploring is the use of TMTX with leucovorin protection, which may allow selective cell kill of MTX-resistant cells without significant toxicity.38
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FOOTNOTES |
Submitted July 26, 1996;
accepted September 25, 1996.
Supported by American Cancer Society Grant No. BC-561C, the Parker-Hughes Foundation, the Charles A. Dana Foundation, the Medical Research Council of Canada (W.F.F.), and Grant No. CA09512 from the National Cancer Institute. R.G. is the recipient of an American Society of Clinical Oncology Young Investigator Award. J.R.B. is an American Cancer Society Professor.
Address reprint requests to Joseph R. Bertino, MD, Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
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REFERENCES |
1.
Jolivet J,
Cowan KH,
Curt GA,
Clendeninn NJ,
Chabner BA:
The pharmacology and clinical use of methotrexate.
N Engl J Med
309:1094,
1983[Medline]
[Order article via Infotrieve]
2.
Bertino JR:
Ode to methotrexate.
J Clin Oncol
11:5,
1993[Medline]
[Order article via Infotrieve]
3.
Bertino JR,
Goker E:
Drug resistance in acute leukemia.
Leuk Lymph
11:37,
1993
4. Allegra CJ: Antifolates, in Chabner BA, Collins JM (eds): Cancer Chemotherapy: Principles and Practice. Philadelphia, PA, Lippincott, 1990, p 110
5.
Schweitzer BI,
Dicker AP,
Bertino JR:
Dihydrofolate reductase as a therapeutic target.
FASEB J
4:2441,
1990[Abstract]
6.
Goker E,
Waltham M,
Kheradpour A,
Trippett T,
Mazumdar M,
Elisseyeff Y,
Schnieders B,
Steinherz P,
Tan C,
Berman E,
Bertino JR:
Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia and is correlated with p53 gene mutations.
Blood
86:677,
1995[Abstract/Free Full Text]
7.
Trippett T,
Schlemmer S,
Elisseyeff Y,
Goker E,
Wachter M,
Steinherz P,
Tan C,
Berman E,
Wright JE,
Rosowsky A,
Schweitzer B,
Bertino JR:
Defective transport as a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia.
Blood
80:1158,
1992[Abstract/Free Full Text]
8.
Matherly LH,
Taub JE,
Ravindranath Y,
Proefke SA,
Wong SC,
Gimotty P,
Buck S,
Wright JE,
Rosowsky A:
Elevated dihydrofolate reductase and impaired methotrexate transport as elements in methotrexate resistance in childhood acute lymphocytic leukemia.
Blood
85:500,
1995[Abstract/Free Full Text]
9.
Williams FMR,
Flintoff WF:
Isolation of a human cDNA that complements a mutant hamster cell defective in methotrexate uptake.
J Biol Chem
270:2987,
1995[Abstract/Free Full Text]
10.
Moscow JA,
Gong M,
He R,
Sgagias MK,
Dixon KH,
Anzick SL,
Meltzer PS,
Cowan KH:
Isolation of a gene encoding a human reduced folate carrier (RFC1) and analysis of its expression in transport-deficient, methotrexate-resistant human breast cancer cells.
Cancer Res
55:3790,
1995[Abstract/Free Full Text]
11.
Wong SC,
Proefke SA,
Bhushan A,
Matherly LH:
Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells.
J Biol Chem
270:17468,
1995[Abstract/Free Full Text]
12.
Prasad PD,
Ramamoorthy S,
Leibach FH,
Ganapathy V:
Molecular cloning of the human placental folate transporter.
Biochem Biophys Res Commun
206:681,
1995[Medline]
[Order article via Infotrieve]
13.
Gaynon PS,
Steinherz PG,
Bleyer AW,
Ablin AR,
Albo VC,
Finkelstein JZ,
Grossman NJ,
Novak LJ,
Pysemany AF,
Reaman GH,
Chappell RJ,
Sather HN,
Hammond GD:
Improved therapy for children with acute lymphoblastic leukemia and unfavorable presenting features: A follow-up report of the Children's Cancer Group study CCG-106.
J Clin Oncol
11:2234,
1993[Abstract/Free Full Text]
14.
Steinherz PG,
Redner A,
Steinherz L,
Meyers P,
Tan C,
Heller G:
Development of a new intensive therapy for acute lymphoblastic leukemia in children at increased risk of early relapse.
Cancer
72:3120,
1993[Medline]
[Order article via Infotrieve]
15.
Berman E,
Weiss M,
Kempin S,
Gee T,
Bertino J,
Clarkson B:
Intensive therapy for adult acute lymphoblastic leukemia.
Semin Hematol
28:72,
1991[Medline]
[Order article via Infotrieve]
16.
Rosowsky A,
Wright JE,
Shapiro H,
Beardsley GP,
Lazarus H:
A new fluorescent dihydrofolate reductase probe for studies of methotrexate resistance.
J Biol Chem
257:14162,
1982[Abstract/Free Full Text]
17.
Rosowsky A,
Wright JE,
Cucchi CA,
Boeheim K,
Frei E III:
Transport of a fluorescent antifolate by methotrexate-sensitive and methotrexate-resistant human leukemic lymphoblasts.
Biochem Pharmacol
35:356,
1986[Medline]
[Order article via Infotrieve]
18.
Assaraf YG,
Feder JN,
Sharma RC,
Wright JE,
Rosowsky A,
Shane B,
Schimke RT:
Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.
J Biol Chem
267:5776,
1992[Abstract/Free Full Text]
19.
Thomas PS:
Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.
Proc Natl Acad Sci USA
77:5201,
1980[Abstract/Free Full Text]
20.
Laborda J:
36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO.
Nucleic Acids Res
19:3998,
1991[Free Full Text]
21.
Horikoshi T,
Danenberg KD,
Stadlbauer THW,
Volkenandt M,
Shea LCC,
Aigner K,
Gustavsson B,
Leichman L,
Frosing R,
Ray M,
Gibson NW,
Spears CP,
Danenberg PV:
Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.
Cancer Res
52:108,
1992[Abstract/Free Full Text]
22.
Johnston PG,
Lenz HJ,
Leichman CG,
Danenberg KD,
Allegra CJ,
Danenberg PV,
Leichman L:
Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors.
Cancer Res
55:1407,
1995[Abstract/Free Full Text]
23. Fleiss JL: Statistical Methods for Rates and Proportions (ed 2). New York, NY, Wiley Interscience, 1981
24.
McCloskey DE,
McGuire JJ,
Russell CA,
Rowan BG,
Bertino JR,
Pizzorno G,
Mini E:
Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines.
J Biol Chem
266:6181,
1991[Abstract/Free Full Text]
25.
Spinella MJ,
Brigle KE,
Sierra EE,
Goldman ID:
Distinguishing between folate receptor mediated transport and reduced folate carrier mediated transport in L1210 leukemia cells.
J Biol Chem
270:7842,
1995[Abstract/Free Full Text]
26.
Westerhof GR,
Rijnbout S,
Schornagel JH,
Pinedo HM,
Peters GJ,
Jansen G:
Functional activity of the reduced folate carrier in KB, MA104 and IGROV-1 cells expressing folate binding protein.
Cancer Res
55:3996,
1995[Abstract/Free Full Text]
27.
Williams FMR,
Murray RC,
Underhill TM,
Flintoff WF:
Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake.
J Biol Chem
269:5810,
1994[Abstract/Free Full Text]
28.
Dixon KH,
Lanpher BC,
Chiu J,
Kelley K,
Cowan KH:
A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells.
J Biol Chem
269:17,
1994[Abstract/Free Full Text]
29.
Klohs WD,
Steinkampf RW,
Besserer JA,
Fry DW:
Cross-resistance of pleiotropically resistant P388 leukemia cells to the lipophilic antifolates trimetrexate and BW 301U.
Cancer Lett
31:253,
1986[Medline]
[Order article via Infotrieve]
30.
Assaraf YG,
Molina A,
Schimke RT:
Cross-resistance to the lipid-soluble antifolate trimetrexate in human carcinoma cells with the multidrug-resistant phenotype.
J Natl Cancer Inst
81:290,
1989[Abstract/Free Full Text]
31.
Spencer HT,
Sorrentino BP,
Pui CH,
Chunduru SK,
Sleep SE,
Blakley RL:
Mutations in the gene for human dihydrofolate reductase: An unlikely cause of clinical relapse in pediatric leukemia after therapy with methotrexate.
Leukemia
10:439,
1996[Medline]
[Order article via Infotrieve]
32. Connolly T, Myers TG, Paull KD, Cowan KH, Moscow JA: RFC1 expression and resistance to methotrexate and trimetrexate in transfected and non-selected cell lines. Proc Am Assoc Cancer Res 37:498, 1996 (abstr)
33.
Brigle KE,
Spinella MJ,
Sierra EE,
Goldman ID:
Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line.
J Biol Chem
270:22974,
1995[Abstract/Free Full Text]
34.
Lin JT,
Tong WP,
Trippett TM,
Niedzwiecki D,
Tao Y,
Tan C,
Steinherz P,
Schweitzer BI,
Bertino JR:
Basis for natural resistance to methotrexate in human acute non-lymphocytic leukemia.
Leuk Res
15:1191,
1991[Medline]
[Order article via Infotrieve]
35.
Goker E,
Kheradpour A,
Waltham M,
Banerjee D,
Tong WP,
Elisseyeff Y,
Bertino JR:
Acute monocytic leukemia: A myeloid subset that may be sensitive to methotrexate.
Leukemia
9:274,
1995[Medline]
[Order article via Infotrieve]
36.
Zochbauer S,
Gaur A,
Brunner R,
Kyrle PA,
Lechner K,
Pirker R:
P-glycoprotein expression as unfavorable prognostic factor in acute myeloid leukemia.
Leukemia
8:974,
1994[Medline]
[Order article via Infotrieve]
37.
Paietta A,
Andersen J,
Racevskis J,
Gallagher R,
Bennett J,
Yunis J,
Cassileth P,
Wiernik PH:
Significantly lower p-glycoprotein expression in acute promyelocytic leukemia than in other types of acute myeloid leukemia: Immunological, molecular and functional analyses.
Leukemia
8:968,
1994[Medline]
[Order article via Infotrieve]
38.
Lacerda JF,
Goker E,
Kheradpour A,
Dennig D,
Elisseyeff Y,
Jagiello C,
O'Reilly RJ,
Bertino JR:
Selective treatment of SCID mice bearing methotrexate-transport resistant human acute leukemia tumors with trimetrexate and leucovorin protection.
Blood
85:2675,
1995[Abstract/Free Full Text]
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D. French, W. Yang, C. Cheng, S. C. Raimondi, C. G. Mullighan, J. R. Downing, W. E. Evans, C.-H. Pui, and M. V. Relling
Acquired variation outweighs inherited variation in whole genome analysis of methotrexate polyglutamate accumulation in leukemia
Blood,
May 7, 2009;
113(19):
4512 - 4520.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Zhao, A. Qiu, E. Tsai, M. Jansen, M. H. Akabas, and I. D. Goldman
The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier
Mol. Pharmacol.,
September 1, 2008;
74(3):
854 - 862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. von Stackelberg, R. Hartmann, C. Buhrer, R. Fengler, G. Janka-Schaub, A. Reiter, G. Mann, K. Schmiegelow, R. Ratei, T. Klingebiel, et al.
High-dose compared with intermediate-dose methotrexate in children with a first relapse of acute lymphoblastic leukemia
Blood,
March 1, 2008;
111(5):
2573 - 2580.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. Mishra, R. Humeniuk, P. J. Mishra, G. S. A. Longo-Sorbello, D. Banerjee, and J. R. Bertino
A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance
PNAS,
August 14, 2007;
104(33):
13513 - 13518.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bhojwani, H. Kang, N. P. Moskowitz, D.-J. Min, H. Lee, J. W. Potter, G. Davidson, C. L. Willman, M. J. Borowitz, I. Belitskaya-Levy, et al.
Biologic pathways associated with relapse in childhood acute lymphoblastic leukemia: a Children's Oncology Group study
Blood,
July 15, 2006;
108(2):
711 - 717.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. L. Gomez, S. L. Santillana, C. S. Vallejos, R. Velarde, J. Sanchez, X. Wang, N. L. Bauer, R. D. Hockett, V. J. Chen, C. Niyikiza, et al.
A Phase II Trial of Pemetrexed in Advanced Breast Cancer: Clinical Response and Association with Molecular Target Expression
Clin. Cancer Res.,
February 1, 2006;
12(3):
832 - 838.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Rothem, B. Berman, M. Stark, G. Jansen, and Y. G. Assaraf
The Reduced Folate Carrier Gene Is a Novel Selectable Marker for Recombinant Protein Overexpression
Mol. Pharmacol.,
September 1, 2005;
68(3):
616 - 624.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J Wolf, T Stranzl, M Filipits, G Pohl, R Pirker, B Leeb, and J S Smolen
Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis
Ann Rheum Dis,
April 1, 2005;
64(4):
564 - 568.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. M. Spees, T. P.F. Gade, G. Yang, W. P. Tong, W. G. Bornmann, R. Gorlick, and J. A. Koutcher
An 19F Magnetic Resonance-Based In Vivo Assay of Solid Tumor Methotrexate Resistance: Proof of Principle
Clin. Cancer Res.,
February 15, 2005;
11(4):
1454 - 1461.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Flatley, S. G. Payton, J. W. Taub, and L. H. Matherly
Primary Acute Lymphoblastic Leukemia Cells Use a Novel Promoter and 5'Noncoding Exon for the Human Reduced Folate Carrier That Encodes a Modified Carrier Translated from an Upstream Translational Start
Clin. Cancer Res.,
August 1, 2004;
10(15):
5111 - 5122.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Rothem, M. Stark, Y. Kaufman, L. Mayo, and Y. G. Assaraf
Reduced Folate Carrier Gene Silencing in Multiple Antifolate-resistant Tumor Cell Lines Is Due to a Simultaneous Loss of Function of Multiple Transcription Factors but Not Promoter Methylation
J. Biol. Chem.,
January 2, 2004;
279(1):
374 - 384.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. DeAngelis
Primary Central Nervous System Lymphoma: A Curable Brain Tumor
J. Clin. Oncol.,
December 15, 2003;
21(24):
4471 - 4473.
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. M. Spees, G. Yang, D. Veach, M. B. Rubio, J. A. Koutcher, and W. Bornmann
A fluorine-labeled methotrexate as a probe for monitoring tumor antifolate pharmacokinetics: Synthesis, in vitro cytotoxicity, and pilot in vivo 19F magnetic resonance spectra
Mol. Cancer Ther.,
October 1, 2003;
2(10):
933 - 939.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Yang, R. Sowers, B. Mazza, J. H. Healey, A. Huvos, H. Grier, M. Bernstein, G. P. Beardsley, M. D. Krailo, M. Devidas, et al.
Sequence Alterations in the Reduced Folate Carrier Are Observed in Osteosarcoma Tumor Samples
Clin. Cancer Res.,
February 1, 2003;
9(2):
837 - 844.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. L. Carroll, D. Bhojwani, D.-J. Min, E. Raetz, M. Relling, S. Davies, J. R. Downing, C. L. Willman, and J. C. Reed
Pediatric Acute Lymphoblastic Leukemia
Hematology,
January 1, 2003;
2003(1):
102 - 131.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Laverdiere, S. Chiasson, I. Costea, A. Moghrabi, and M. Krajinovic
Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukemia
Blood,
November 15, 2002;
100(10):
3832 - 3834.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Sadlish, F. M. R. Williams, and W. F. Flintoff
Functional Role of Arginine 373 in Substrate Translocation by the Reduced Folate Carrier
J. Biol. Chem.,
October 25, 2002;
277(44):
42105 - 42112.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. H. Sarris, A. Phan, M. Duvic, J. Romaguera, P. McLaughlin, O. Mesina, K. King, L. J. Medeiros, G. Z. Rassidakis, B. Samuels, et al.
Trimetrexate in Relapsed T-Cell Lymphoma With Skin Involvement
J. Clin. Oncol.,
June 15, 2002;
20(12):
2876 - 2880.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. D. Goldman
Membrane Transport of Chemotherapeutics and Drug Resistance: Beyond the ABC Family of Exporters to the Role of Carrier-mediated Processes
Clin. Cancer Res.,
January 1, 2002;
8(1):
4 - 6.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Worm, A. F. Kirkin, K. N. Dzhandzhugazyan, and P. Guldberg
Methylation-dependent Silencing of the Reduced Folate Carrier Gene in Inherently Methotrexate-resistant Human Breast Cancer Cells
J. Biol. Chem.,
October 19, 2001;
276(43):
39990 - 40000.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Zeng, Z.-S. Chen, M. G. Belinsky, P. A. Rea, and G. D. Kruh
Transport of Methotrexate (MTX) and Folates by Multidrug Resistance Protein (MRP) 3 and MRP1: Effect of Polyglutamylation on MTX Transport
Cancer Res.,
October 1, 2001;
61(19):
7225 - 7232.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. M. Trippett, S. Garcia, K. Manova, R. Mody, L. Cohen-Gould, W. Flintoff, and J. R. Bertino
Localization of a Human Reduced Folate Carrier Protein in the Mitochondrial as Well as the Cell Membrane of Leukemia Cells
Cancer Res.,
March 1, 2001;
61(5):
1941 - 1947.
[Abstract]
[Full Text]
|
|
|
|
|
|
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R. Zhao, S. Babani, F. Gao, L. Liu, and I. D. Goldman
The Mechanism of Transport of the Multitargeted Antifolate (MTA) and Its Cross-resistance Pattern in Cells with Markedly Impaired Transport of Methotrexate
Clin. Cancer Res.,
September 1, 2000;
6(9):
3687 - 3695.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Zhao, F. Gao, S. Babani, and I. D. Goldman
Sensitivity to 5,10-Dideazatetrahydrofolate Is Fully Conserved in a Murine Leukemia Cell Line Highly Resistant to Methotrexate Due to Impaired Transport Mediated by the Reduced Folate Carrier
Clin. Cancer Res.,
August 1, 2000;
6(8):
3304 - 3311.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. H. Mahoney Jr, J. J. Shuster, R. Nitschke, S. Lauer, C. P. Steuber, and B. Camitta
Intensification With Intermediate-Dose Intravenous Methotrexate Is Effective Therapy for Children With Lower-Risk B-Precursor Acute Lymphoblastic Leukemia: A Pediatric Oncology Group Study
J. Clin. Oncol.,
March 13, 2000;
18(6):
1285 - 1294.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. G. Rots, R. Pieters, G. J. Peters, C. H. van Zantwijk, R. Mauritz, P. Noordhuis, J. C. Willey, K. Hahlen, U. Creutzig, G. Janka-Schaub, et al.
Circumvention of Methotrexate Resistance in Childhood Leukemia Subtypes by Rationally Designed Antifolates
Blood,
November 1, 1999;
94(9):
3121 - 3128.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Zhao, I. G. Sharina, and I. D. Goldman
Pattern of Mutations that Results in Loss of Reduced Folate Carrier Function under Antifolate Selective Pressure Augmented by Chemical Mutagenesis
Mol. Pharmacol.,
July 1, 1999;
56(1):
68 - 76.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. C. Wong, L. Zhang, T. L. Witt, S. A. Proefke, A. Bhushan, and L. H. Matherly
Impaired Membrane Transport in Methotrexate-resistant CCRF-CEM Cells Involves Early Translation Termination and Increased Turnover of a Mutant Reduced Folate Carrier
J. Biol. Chem.,
April 9, 1999;
274(15):
10388 - 10394.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Guo, J. H. Healey, P. A. Meyers, M. Ladanyi, A. G. Huvos, J. R. Bertino, and R. Gorlick
Mechanisms of Methotrexate Resistance in Osteosarcoma
Clin. Cancer Res.,
March 1, 1999;
5(3):
621 - 627.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. M. Belkov, E. Y. Krynetski, J. D. Schuetz, Y. Yanishevski, E. Masson, S. Mathew, S. Raimondi, C.-H. Pui, M. V. Relling, and W. E. Evans
Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A Mechanism for Ploidy but not Lineage Differences in Methotrexate Accumulation
Blood,
March 1, 1999;
93(5):
1643 - 1650.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. G. Rots, R. Pieters, G. J. Peters, P. Noordhuis, C. H. van Zantwijk, G. J.L. Kaspers, K. Hahlen, U. Creutzig, A. J.P. Veerman, and G. Jansen
Role of Folylpolyglutamate Synthetase and Folylpolyglutamate Hydrolase in Methotrexate Accumulation and Polyglutamylation in Childhood Leukemia
Blood,
March 1, 1999;
93(5):
1677 - 1683.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X.-L. Sun, H. N. Jayaram, K. Gharehbaghi, Q.-J. Li, X. Xiao, and A. C. Antony
Modulation of the Cytotoxicity of 3'-Azido-3'-deoxythymidine and Methotrexate after Transduction of Folate Receptor cDNA into Human Cervical Carcinoma: Identification of a Correlation between Folate Receptor Expression and Thymidine Kinase Activity
Cancer Res.,
February 1, 1999;
59(4):
940 - 946.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Jansen, R. Mauritz, S. Drori, H. Sprecher, I. Kathmann, M. Bunni, D. G. Priest, P. Noordhuis, J. H. Schornagel, H. M. Pinedo, et al.
A Structurally Altered Human Reduced Folate Carrier with Increased Folic Acid Transport Mediates a Novel Mechanism of Antifolate Resistance
J. Biol. Chem.,
November 13, 1998;
273(46):
30189 - 30198.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Zhao, Y. G. Assaraf, and I. D. Goldman
A Mutated Murine Reduced Folate Carrier (RFC1) with Increased Affinity for Folic Acid, Decreased Affinity for Methotrexate, and an Obligatory Anion Requirement for Transport Function
J. Biol. Chem.,
July 24, 1998;
273(30):
19065 - 19071.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Zhao, Y. G. Assaraf, and I. D. Goldman
A Reduced Folate Carrier Mutation Produces Substrate-dependent Alterations in Carrier Mobility in Murine Leukemia Cells and Methotrexate Resistance with Conservation of Growth in 5-Formyltetrahydrofolate
J. Biol. Chem.,
April 3, 1998;
273(14):
7873 - 7879.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. S.A. Longo, R. Gorlick, W. P. Tong, E. Ercikan, and J. R. Bertino
Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells
Blood,
August 1, 1997;
90(3):
1241 - 1245.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Zhao, S. Titus, F. Gao, R. G. Moran, and I. D. Goldman
Molecular Analysis of Murine Leukemia Cell Lines Resistant to 5,10-Dideazatetrahydrofolate Identifies Several Amino Acids Critical to the Function of Folylpolyglutamate Synthetase
J. Biol. Chem.,
August 18, 2000;
275(34):
26599 - 26606.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. R. Whetstine and L. H. Matherly
The Basal Promoters for the Human Reduced Folate Carrier Gene Are Regulated by a GC-box and a cAMP-response Element/AP-1-like Element. BASIS FOR TISSUE-SPECIFIC GENE EXPRESSION
J. Biol. Chem.,
February 23, 2001;
276(9):
6350 - 6358.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract