Large diversity in transport‐mediated methotrexate resistance in human leukemia cell line CCRF‐CEM established in a high concentration of leucovorin

Abstract

ethotrexate (MTX), a classical folate antagonist, has been widely used in the treatment of various kinds of human hematological and solid tumors. 1) However, resistance to this drug is a major clinical obstacle to its curative potential. The mechanisms of development of resistance to MTX have been explored in cultured cells. [1][2][3][4] Combinations of an elevated level of the target enzyme, dihydrofolate reductase (DHFR) due to gene amplification, impaired membrane transport of the drug, alteration in DHFR resulting in decreased binding of MTX, reduced polyglutamylation of MTX and a decreased level of thymidylate synthase (TS) have been suggested to be major mechanisms of resistance. To overcome these known mechanisms by which cells become resistant to MTX, highdose MTX regimens have been used in combination with leucovorin (LV) for the rescue of normal cells from MTX cytotoxicity.5) To obtain differential effects of MTX on tumor and normal cells, optimal scheduling of MTX and LV administration is of critical importance.6, 7) High-dose MTX therapy with LV rescue on an optimal administration schedule resulted in a better treatment outcome of many tumors, and has been used as a major regimen of chemotherapy in child acute lymphoblastic leukemia and osteosarcoma. 1,6) However, the continuation of high-dose MTX therapy with LV rescue frequently fails to eradicate MTX-resistant tumor cells, because of the emergence of drug-resistant cells.1) It has been suggested that the excessive use of LV could have potentially deleterious effects by either recruiting tumors to varying degrees or making them refractory to subsequent MTX therapy. 6, 7)Our earlier study suggested an important role of folate in the development of MTX resistance.2) In the presence of a physiological level of a reduced form of folate, LV, MTX-resistant K562 cells emerged with DHFR overexpression due to gene amplification. In contrast, in the presence of an excess of oxidized folate, transport-mediated MTX resistance was established.2) Thus, the presence of a very high level of LV in highdose MTX regimens with LV rescue would affect the establishment of mechanisms of MTX resistance. To elucidate possible mechanisms behind the appearance of refractory tumors to this therapy, we generated MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of an excess of LV. Continuous exposure of cells to MTX resulted in heterogeneous cell clones with a wide disparity of transportmediated MTX resistance over a 4 logarithmic range, associated with differential alterations of the reduced folate carrier (RFC) gene. Such a wide disparity in transport-mediated MTX resistance may partly explain the appearance of refractory tumors after a high-dose MTX regimen with LV rescue, and indicate the importance of the concentration and schedule of LV administration in high-dose MTX regimens. These cell lines may serve as models for investigation of the molecular mechanism(s) behind refractory tumors in high-dose MTX regimens with LV rescue.

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

“…Despite these preventive measures, MTXinduced resistance and toxicity continue to occur, although infrequently. Mechanisms including elevated DHFR, decreased thymidylate synthase, impaired folate/antifolate transportation, and decreased polyglutamylation on MTX have been proposed to contribute to the development of MTX resistance (Asai et al, 2003). It is also postulated that the excessive use of leucovorin makes tumor cells refractory to subsequent MTX therapy (Bleyer, 1977;Sirotnak et al, 1978).…”

The investigation of epsilon toxin effects on different cancerous cell lines and its synergism effect with methotrexate

“…Methotrexate is a classical folate antagonist, which has a wide application in the treatment of various types of solid tumors, either alone or in combination with other chemotherapeutic agents. [24,25] A major problem in cancer therapy is the emergence of tumor drug resistance with MTX. [6,26] Hence, several other strategies have been developed to overcome this problem.…”

Pharmakogenomik

“…Aus Studien geht hervor, dass der bei mehr als 30% der Kaukasier vorkommende TT-Genotyp mit einer signifikant höheren MTX-Toxizität assoziiert ist. Bei den betroffenen Patienten ist daher eine Dosisanpassung des Zytostatikums indiziert [4,27].Als mögliche Ursache eines Therapieversagens unter Hochdosis-MTX-Therapie (MTX-Resistenz) konnte eine gesteigerte mRNS-Expression des Dihydrofolatreduktasegens und eine verminderte Expression des Thymidylatsynthasegens, aber keine Verän-derung im Folattransporter(RFC1)-Gen identifiziert werden [1]. …”