5-Amino-4-Imidazolecarboxamide Riboside Potentiates Both Transport of Reduced Folates and Antifolates by the Human Reduced Folate Carrier and Their Subsequent Metabolism

Abstract

Transport is required before reduced folates and anticancer antifolates [e.g., methotrexate (MTX)] exert their physiologic functions or cytotoxic effects. The folate/antifolate transporter with the widest tissue distribution and greatest activity is the reduced folate carrier (RFC). There is little evidence that RFCmediated influx is posttranscriptionally regulated. We show that [3 H]MTX influx in CCRF-CEM human childhood T-leukemia cells is potentiated up to 6-fold by exogenous 5-amino-4-imidazolecarboxamide riboside (AICAr) in a AICAr and MTX concentration-dependent manner. Metabolism to more biologically active polyglutamate forms is also potentiated for MTX and other antifolates. That potentiation of influx by AICAr is mediated by effects on the RFC is supported by analyses FAICAr showing (a) similarity and magnitude of kinetic constants for [ 3 H]MTX influx; (b) similarity of inhibitory potency of known RFC substrates; (c) lack of potentiation in a CCRF-CEM subline that does not express the RFC; and (d) similarity of time and temperature dependence. Potentiation occurs rapidly and does not require new protein synthesis. Effects of specific inhibitors of folate metabolism and the time and sequence of AICAr incubation with cells suggest that both dihydrofolate reductase inhibition and metabolism of AICAr are essential for potentiation. Acute folate deficiency or incubation of CCRF-CEM with AICArrelated metabolites (e.g., adenosine) does not initiate potentiation. AICAr increases growth inhibitory potency of MTX and aminopterin against CCRF-CEM cells when both AICAr and antifolate are present for the first 24 hours of a 120-hour growth period. AICAr is the first small molecule that regulates RFC activity. (Cancer Res 2006; 66(7): 3836-44)

Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

“…There are some differences between these findings in HeLa cells and what was reported earlier in studies with CCRF-CEM human T leukemia cells (McGuire et al, 2006). For instance, stimulation of MTX influx by AICAR was delayed even when cells were pretreated with this agent, becoming instantaneous when cells were pretreated with trimetrexate, a lipophilic DHFR inhibitor.…”

“…For instance, stimulation of MTX influx by AICAR was delayed even when cells were pretreated with this agent, becoming instantaneous when cells were pretreated with trimetrexate, a lipophilic DHFR inhibitor. Likewise, augmentation of net 5-formyl-THF uptake by AICAR only occurred if cells were pretreated with trimetrexate, leading the authors to suggest that inhibition of tetrahydrofolate generation is a conditio sine qua non for AICAR potentiation (McGuire et al, 2006). However, in the current study, augmentation of 5-formyl-THF and of 5-methyl-THF and MTX, initial rates occurred without any prior perturbation of DHFR activity.…”

“…In this report, we expand the understanding of the mechanistic basis for the previously reported impact of AICAR on folate and antifolate uptake by kinetic analyses and assessment of AICAR metabolism within cells (McGuire et al, 2006). Augmentation of folate transport in HeLa cells by AICAR is due to the following: 1) AICAR transport into cells, 2) the formation of ZMP from AICAR within cells, and 3) inhibition by ZMP of folate efflux and trans-stimulation of folate influx that together produce a substantial augmentation of net folate uptake.…”

“…5-Aminoimidazole-4-carboxamide riboside (AICAR) was shown to enhance MTX and 5-formyltetrahydrofolate uptake into CCRF-CEM T leukemia cells (McGuire et al, 2006). This effect was attributed to the salutary impact of AICAR on RFC function (McGuire et al, 2006).…”

“…This effect was attributed to the salutary impact of AICAR on RFC function (McGuire et al, 2006). However, the mechanism by which this occurred was not established nor was the possibility that the enhanced net transport of folates might be a result of inhibition of ATP-binding cassette exporters excluded.…”