Cytotoxicity of methotrexate and trimetrexate and its reversal by folinic acid in human leukemic CCRF-CEM cells with carrier-mediated and receptor-mediated folate uptake

Veer, Linda J. van der; Westerhof, G R; Rijksen, Gert; Schornagel, J. H.; Jansen, Gerrit

doi:10.1016/0145-2126(89)90005-2

Cited by 19 publications

(8 citation statements)

References 14 publications

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“…Previous studies have indicated that modulation of RFC activity has opposite effects on MTX and TMTX cytotoxicity (Ohnuma et al, 1985;van der Veer et al, 1989). The RFC1-transfection studies demonstrate that reconstitution of RFC1 activity can change the relative resistance to MTX and TMTX by between 2 and 3 orders of magnitude in a MTX R ZR-75-1 cell line that is null for both RFC and FR activity.…”

Section: Resultsmentioning

confidence: 93%

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

et al. 1997

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Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI 50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTX R ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFC1 RNA levels by a quantitative RT-PCR assay. RFC1 RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTX R ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines. Int.

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Section: Resultsmentioning

confidence: 93%

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

et al. 1997

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“…The human KB nasopharyngeal epidermoid line with high-level expression of FR-a is highly sensitive to MTX, and transport is mediated largely by this process (Deutsch et al, 1989). On the other hand, CCRF-CEM human leukemia cells that lack RFC but have increased FR-a expression have markedly diminished transport of MTX relative to wild-type cells and are highly resistant to the drug ( Van der Veer et al, 1989). In the case of folic acid with high affinity for FRs but low affinity for RFC, the level of FR expression is a major determinant of the rate of transport and the receptor is upregulated by growth in media with low folate levels (Henderson et al, 1988;Jansen et al, 1989).…”

Section: Folic Acid Receptor-mediated Transportmentioning

confidence: 99%

Resistance to antifolates

2003

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“…33,34 Second, tumor cells differ from normal cells in their transport properties. [35][36][37][38][39] Results from Chello et al 40 showed, for example, a 30-fold higher affinity for methotrexate in the mouse leukemia cell line L1210 than in freshly isolated murine intestinal cells.Hepatocytes are exceptional with respect to folate transport. In contrast to most other cells, hepatocytes express a separate carrier system for 5-methyltetrahydrofolate, which is different from the reduced folate transporter.…”

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confidence: 99%

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

Honscha¹,

Dötsch²,

Thomsen³

et al. 2000

Hepatology

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The antifolates methotrexate and trimetrexate are potent inhibitors of the dihydrofolate reductase, an enzyme that delivers by the metabolism of folate and its reduced forms the C1-groups for the synthesis of nucleotides and amino acids. Methotrexate, which has a comparatively moderate toxicity is one of the most important clinically used chemotherapeutics for the treatment of leukemias, such as acute lymphoblastic leukemia of children. It is also used in breast cancer, squamous tumors of the head and neck, lymphomas, and choriocarcinomas. 1,2 Uptake studies of methotrexate and the natural folic acid derivatives in different tumor and normal cells have shown that at least two different carrier systems mediate the uptake of these compounds. The 40 kd folate binding protein has a high affinity for folic acid but a low affinity for reduced folates. [3][4][5][6][7][8][9][10] The corresponding complementary DNAs (cDNAs) as well as genomic clones have been isolated from various sources, 11-19 but a detailed characterization of the transporter function is still missing.Different cDNAs and genomic clones for the reduced folate carrier, which has a high affinity for the reduced folates 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but a low affinity for folate [20][21][22][23][24] have been also isolated from different immortal cell lines 25-31 but the clones were not characterized in detail.The expression and the abundance of the two carriers varied in different tumor and normal cells. From uptake studies it became evident that, first, in some cell systems both transport systems work in tandem 32 whereas two independently operating carrier systems were also described. 33,34 Second, tumor cells differ from normal cells in their transport properties. [35][36][37][38][39] Results from Chello et al. 40 showed, for example, a 30-fold higher affinity for methotrexate in the mouse leukemia cell line L1210 than in freshly isolated murine intestinal cells.Hepatocytes are exceptional with respect to folate transport. In contrast to most other cells, hepatocytes express a separate carrier system for 5-methyltetrahydrofolate, which is different from the reduced folate transporter. 41,42 We recently characterized the uptake of methotrexate into freshly isolated hepatocytes 43 and showed that this chemotherapeutic is taken up by a sodium-dependent active transport system, which is inhibited by the reduced folates, dihydrofolate, and tetrahydrofolate but not by folate itself. Moreover, substrates of two well-known organic anion transport systems in the liver, the Ntcp1 44,45 and the oatp1, 46 like the bile acids taurocholate and cholate as well as xenobiotics such as bromosulfophthalein, the loop diuretic bumetanide, and the mycotoxin ochratoxin A are potent inhibitors of the hepatocellular methotrexate carrier. Because some substrates were Abbreviations: cDNA, complementary DNA; RL-Mtx, rat liver methotrexate transporter; RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; DMEM, Dulbecco modi...

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Cytotoxicity of methotrexate and trimetrexate and its reversal by folinic acid in human leukemic CCRF-CEM cells with carrier-mediated and receptor-mediated folate uptake

Cited by 19 publications

References 14 publications

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

Resistance to antifolates

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

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