Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of sequence variants in the human reduced folate carrier (hRFC) was assessed in leukemia blasts from children with acute lymphoblastic leukemia (ALL) and in normal peripheral blood specimens. A CATG frame shift insertion at position 191 was detected in 10-60% of hRFC transcripts from 10 of 16 ALL specimens, by RFLP analysis and direct sequencing of hRFC cDNAs. In genomic DNAs prepared from 105 leukemia (n = 54) and non-leukemia (n = 51) specimens, PCR amplifications and direct sequencing of exon 3 identified a high-frequency G to A single nucleotide polymorphism at position 80 that resulted in a change of arginine-27 to histidine-27. The allelic frequencies of G/A80 were nearly identical for the non-leukemia (42.2% CGC and 57.8% CAC) and leukemia (40.7% CGC and 59.3% CAC) genomic DNAs. In cDNAs prepared from 10 of these ALL patients, identical allelic frequencies (40 and 60%, respectively) were recorded. In up to 62 genomic DNAs, hRFC-coding exons 4-7 were PCR-amplified and sequenced. A high-abundance C/T696 polymorphism was detected with nearly identical frequencies for both alleles, and a heterozygous C/A1242 sequence variant was identified in two ALL specimens. Both C/T696 and C/A1242 were phenotypically silent. In transport assays with [(3)H]methotrexate and [(3)H]5-formyl tetrahydrofolate, nearly identical uptake rates were measured for the arginine-27- and histidine-27-hRFC proteins expressed in transport-impaired K562 cells. Although there were no significant differences between the kinetic parameters for methotrexate transport for the hRFC forms, minor (approximately 2-fold) differences were measured in the K(i)s for other substrates including Tomudex, 5,10-dideazatetrahydrofolate, GW1843U89, and 10-ethyl-10-deazaaminopterin and for 5-formyl tetrahydrofolate.
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PMID:Single nucleotide polymorphisms in the human reduced folate carrier: characterization of a high-frequency G/A variant at position 80 and transport properties of the His(27) and Arg(27) carriers. 1170 57

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.
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PMID:Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis. 1184 83

We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates that target various folate-dependent enzymes. These include the dihydrofolate reductase (DHFR) inhibitors edatrexate, methotrexate and aminopterin, the thymidylate synthase (TS) inhibitors ZD1694 and GW1843, the glycinamide ribonucleotide formyltransferase (GARTF) inhibitor DDATHF as well as the multitargeted antifolate LY231514 inhibiting both TS, DHFR and GARTF. Fourteen antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired folylpoly-gamma-glutamate synthetase (FPGS) activity as these cell lines: 1) typically lost 90-99% of parental FPGS activity; 2) expressed 1.4-3.3-fold less FPGS mRNA (only 4 cell lines); 3) displayed up to 10(5)-fold resistance to polyglutamylation-dependent antifolates including ZD1694 and MTA; 4) retained sensitivity to polyglutamylation-independent antifolates including ZD9331 and PT523; 5) were up to 19-fold hypersensitive to the lipid-soluble antifolates trimetrexate and AG377; 6) had a normal or a small decrease in [(3)H]MTX transport; and 7) had a 2.1-8.3-fold decreased cellular folate pools and a consequently increased folate growth requirement. The remaining 3 antifolate-resistant sublines lost 94-97% of parental [(3)H]MTX transport and thus displayed a high level resistance to all hydrophilic antifolates. To screen for mutations in the hFPGS gene, we devised an RT-PCR single strand conformational polymorphism (SSCP) assay. RT-PCR-SSCP analysis and DNA sequencing showed that only a single FPGS-deficient subline harbored an FPGS mutation (Cys346Phe). Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within alpha-helix A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent antifolates but in the same time retain parental sensitivity or hypersensitivity to polyglutamylation-independent antifolates or lipophilic antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.
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PMID:Loss of folylpoly-gamma-glutamate synthetase activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates in multiple human leukemia sublines. 1249 65

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.
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PMID:Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line. 1266 51

Antifolates are the oldest of the antimetabolite class of anticancer agents and were one of the first modern anticancer drugs. The first clinically useful antifolate, described in 1947, was 2,4-diamino-pteroylglutamate (4-amino-folic acid; aminopterin; AMT) which yielded the first-ever remissions in childhood leukemia. AMT was soon superseded by its 10-methyl congener, methotrexate (MTX), based on toxicity considerations; MTX remains, with one limited exception, the only antifolate anticancer agent in clinical use to this date. Because of the safety and utility of MTX, considerable effort has been invested in attempting to design more therapeutically selective antifolates or antifolates with a wider tumor spectrum. Initially, the design was based on the burgeoning knowledge of folate-dependent pathways and the determinants of the mechanism of action of MTX. These determinants include transport, the tight-binding inhibition of its target (the folate-dependent enzyme dihydrofolate reductase (DHFR)), and metabolism of MTX to poly-gamma-glutamate (Glu(n)) metabolites. These early studies led to the development of other antifolate DHFR inhibitors of two types: (1). "classical" analogs that use the same cellular transport systems as MTX and are also metabolized to Glu(n); and (2). "nonclassical" (i.e., lipophilic) analogs that do not require transport systems and that are not metabolized to Glu(n). Although several of these analogs have undergone clinical trial, none is proved superior to MTX. Detailed examination of the mechanisms of cytotoxicity and selectivity of MTX showed that inhibition of both dTMP synthesis and de novo purine synthesis, secondary to DHFR inhibition, led to DNA synthesis inhibition and subsequent cell death; inhibition of other folate-dependent pathways did not appear necessary for cell death. Further studies showed that the contribution of inhibition of dTMP or purine synthesis to cell death varied in different cell types. These data suggested that inhibition of one of these pathways individually might (at least in some cases) be therapeutically superior to the dual inhibition induced by MTX. Thus in rational design and in structure-based design studies, two new classes of antifolate enzyme inhibitors were elaborated-direct inhibitors of thymidylate synthase (TMPS) and direct inhibitors of one or both of the two folate-dependent enzymes of de novo purine synthesis. Members of each class included both classical and nonclassical types. After preclinical evaluation, several of these have moved into clinical trials. To date only one new TMPS inhibitor has successfully completed clinical trials and been approved for routine use; this drug, Tomudex (D1694, raltitrexed) is currently approved only in Europe and only for the treatment of colon cancer. This still represents a step forward for antifolates, however, since MTX is well-known to be ineffective in colon cancer; thus Tomudex extends the tumor range of antifolates. Antifolate development continues. Based on the immense body of knowledge now extant on antifolates, specific aspects of the mechanism of action have been the focus. Newer antifolates have been described that inhibit more than one pathway in folate metabolism, that have improved delivery, or that inhibit other targets in folate metabolism. These new analogs are in various stages of preclinical and clinical development.
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PMID:Anticancer antifolates: current status and future directions. 1452 44


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