Effects of methotrexate esters and other lipophilic antifolates on methotrexate-resistant human leukemic lymphoblasts

Rosowsky, A.; Lazarus, Herbert; Yuan, Grace C.; Beltz, William R.; Mangini, Lynn; Abelson, Herbert T.; Modest, Edward J.; Frei, Emil

doi:10.1016/0006-2952(80)90391-3

Cited by 86 publications

(65 citation statements)

References 16 publications

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“…Prolonged retention of MTXPG and inhibition of DNA synthesis in the absence of free drug, which disappears from plasma within 24 h for most conventional dose regimens, could result from this process. The variability in the capacity of these three breast cancer cell lines to form MTXPG further suggests that this process, as well as drug transport (29) and target enzyme concentration (30), may be an important determinant of de novo or acquired resistance to MTX.…”

Section: Methodsmentioning

confidence: 99%

Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

Jolivet¹,

Schilsky²,

Bailey³

et al. 1982

J. Clin. Invest.

163

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A B S T R A C T To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines converted the drug to y-polyglutamate derivatives in a dose-and timedependent reaction. After 24-h incubations with 2 zM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/ g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH2-10-CH3-PteGlu5 and 47 and 38% of the 4-NH2-10-CH3-PteGlu4 remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. MTX and an additional 24 h in drug-free medium,[3H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [3H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 ,uM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 jM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates.These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

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Section: Methodsmentioning

confidence: 99%

Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

Jolivet¹,

Schilsky²,

Bailey³

et al. 1982

J. Clin. Invest.

163

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“…Seven CCRF-CEM sublines with different transport characteristics were grown in RPMI-medium in 5% CO 2 and harvested at logarithmic phase. [26][27][28][29] Leukemia RNA extraction and reverse transcriptase reaction RNA isolation was according to manufacturer's directions except for a repetition of the chloroform-phenol extraction, and an extension of the precipitation step in isopropanol at −20°C for at least overnight. A PCR reaction was performed on each RNA sample using ␤-actin primers situated in exons 3 and 4 (Table 1) to determine possible genomic DNA contamination.…”

Section: Patient Samples and Cell Linesmentioning

confidence: 99%

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

et al. 2000

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Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of ␤-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three-and four-fold higher, respectively; P Ͻ 0.001), while FPGS expression was lower (three-fold, P ‫؍‬ 0.006) compared to common/preB-ALL. The ratio of (DHFR ؋ FPGH)/(RFC ؋ FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P Ͻ 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P ‫؍‬ 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (fourfold higher; P ‫؍‬ 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r ‫؍‬ 0.80; P Ͻ 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance. Leukemia (2000) 14, 2166-2175.

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“…RFC1-mediated transport is essential for the activity of most folate-based chemotherapeutic drugs, such as methotrexate (MTX), along with new-generation inhibitors of tetrahydrofolate cofactor-dependent enzymes that undergo polyglutamylation at the ␥-carboxyl position of the glutamate moiety (Jackman and Calvert, 1995;Mendelsohn et al, 1996;Shih et al, 1998). Impaired membrane transport is a common mechanism of acquired resistance to these drugs in both murine and human model tumor systems and in human neoplasms after treatment of patients with MTX (Rosowsky et al, 1980;Sirotnak et al, 1981Sirotnak et al, , 1985Cowan and Jolivet, 1984;Schuetz et al, 1988;Underhill and Flintoff, 1989;Pui and Evans, 1998). This has been studied in detail as an important factor in treatment failure in patients with acute lymphoblastic leukemia (Trippett et al, 1992;Matherly et al, 1995;Gorlick et al, 1997;Zhang et al, 1998) With the recent cloning of the RFC1 gene from a variety of species, an understanding of the molecular basis for transport-related drug resistance has now become possible.…”

mentioning

confidence: 99%

Pattern of Mutations that Results in Loss of Reduced Folate Carrier Function under Antifolate Selective Pressure Augmented by Chemical Mutagenesis

1999

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Chemical mutagenesis with N-methyl-N-nitrosourea was employed to study the pattern of mutations in the reduced folate carrier (RFC1) that results in transport-related methotrexate resistance and to identify amino acid residues that are critical to carrier structure and/or function. Thirty-four methotrexate transport-defective L1210 leukemia cell lines were isolated with folic acid as the sole folate source under antifolate selective pressure. The RFC1 mRNA levels were comparable with, or not substantially decreased, in most of these cell lines relative to wild-type L1210 cells. The molecular basis for the transport defects was investigated by sequencing multiple RFC1 cDNA clones isolated from these mutants by reverse transcriptionpolymerase chain reaction, which encompassed the entire coding region. The mutations identified were further confirmed either by direct sequencing or, when applicable, by restriction analysis of total reverse transcription-polymerase chain reaction products. The majority of mutations (21) led to single amino acid substitutions that were in, or near, 9 of 12 predicted transmembrane domains, with the highest frequencies in the first, fifth, and eighth. There were no mutations in the sixth, ninth, and twelfth transmembrane domains. Glycine, serine, and arginine were the most frequently mutated residues. These data suggest that several transmembrane domains, rather than the amino-and carboxyl-termini, and the large intracellular loop between the sixth and seventh transmembrane domains play key roles as sites for RFC1 inactivation because of single point mutations. This panel of mutated cell lines offers an important resource for studies on RFC1 structure-function and for the evaluation of transport-related cross-resistance patterns with new-generation antifolate inhibitors of tetrahydrofolate cofactor-dependent enzymes.

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Effects of methotrexate esters and other lipophilic antifolates on methotrexate-resistant human leukemic lymphoblasts

Cited by 86 publications

References 16 publications

Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Pattern of Mutations that Results in Loss of Reduced Folate Carrier Function under Antifolate Selective Pressure Augmented by Chemical Mutagenesis

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