Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

Sirotnak, Francis M.; Chello, Paul L.; Moccio, D. M.; Kisliuk, Roy L.; Combépine, G.; Gaumont, Y.; Montgomery, John A.

doi:10.1016/0006-2952(79)90599-9

Cited by 71 publications

(15 citation statements)

References 15 publications

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“…Concentrations for L-5-formyltetrahydrofolate and L-5-methyltetrahydrofolate are expressed as the natural (/) diastereosisomer. Previous studies [21] from our laboratory have shown that the high-affinity carrier system in intact L1210 cells exhibited during influx, stereospecificity at carbon 6 of L-5-formyltetrahydrofolate. From data on competition experiments with both natural and unnatural (d) isomers of 5-formyltetrahydrofolate reported at that time, we would conclude that uptake of the unnatural isomer by this system could amount to less than 4% of the total carrier-mediated uptake.…”

Section: Methodsmentioning

confidence: 86%

Relationships between carrier-mediated transport of folate compounds by L1210 leukemia cells: Evidence for multiplicity of entry routes with different kinetic properties expressed in plasma membrane vesicles

1983

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Various independent kinetic criteria for indicating multiplicity of carrier-mediated entry of folate compounds into L1210 cell plasma membrane vesicles are studied. We find a marked inconsistency between values for influx Km and Ki in reciprocal experiments measuring competition between various folate compounds as well as inconsistent effects of transloading shown for 5-formyltetrahydrofolate influx, but not folic acid influx. These results argue strongly against a one-carrier model for transport of folate compounds. The most straightforward interpretation of our data is that two distinct transport systems mediate entry of folate compounds in L1210 plasma membrane vesicles. If a two-carrier model is correct, then our data indicate that one of the carriers has low capacity and high affinity for folate coenzymes and methotrexate. This system is apparently negligible as a transport route for folic acid. Transtimulation of initial influx by substrates of the low capacity system is obtained following transloading with coenzymes but not by transloading with folic acid. Our data indicate that the second folate transport system postulated by the two-carrier model has a low affinity for all the folate compounds studied. Nevertheless, the putative second system is significant, especially for folic acid transport, because it has a much higher capacity than the first transport system. In contrast to the first system, transloading with any of the folate compounds studied had no effect on initial influx mediated by the second folate transport system. The two systems are also differentially inhibited by pCMBS, DIDS and SITS and the influx Vmax for the high-affinity/low-capacity system was altered in a vesicle preparation derived from a methotrexate resistant L1210 cell line.

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Section: Methodsmentioning

confidence: 86%

Relationships between carrier-mediated transport of folate compounds by L1210 leukemia cells: Evidence for multiplicity of entry routes with different kinetic properties expressed in plasma membrane vesicles

1983

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“…Transport of (6S)5-formyl THF is preferred over the (6R) stereoisomer (Sirotnak et al, 1979), although transport is not stereospecific for 5-methyl THF (White et al, 1978;Chello et al, 1982). Whereas 5-methyl and 5-formyl THF both show low micromolar affinities for RFC, folic acid is a poor RFC substrate with binding affinities one to two orders of magnitude less than those for the reduced folate forms (Goldman et al, 1968;Westerhof et al, 1995).…”

Section: Biology Of Rfcmentioning

confidence: 99%

The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

2014

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This review summarizes the biology of the major facilitative membrane transporters, the reduced folate carrier (RFC) (Solute Carrier 19A1) and the proton-coupled folate transporter (PCFT) (Solute Carrier 46A1). Folates are essential vitamins, and folate deficiency contributes to a variety of health disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates the intestinal absorption of dietary folates and appears to be important for transport of folates into the central nervous system. Clinically relevant antifolates for cancer, such as methotrexate and pralatrexate, are transported by RFC, and loss of RFC transport is an important mechanism of methotrexate resistance in cancer cell lines and in patients. PCFT is expressed in human tumors, and is active at pH conditions associated with the tumor microenvironment. Pemetrexed is an excellent substrate for both RFC and PCFT. Novel tumor-targeted antifolates related to pemetrexed with selective membrane transport by PCFT over RFC are being developed. In recent years, there have been major advances in understanding the structural and functional properties and the regulation of RFC and PCFT. The molecular bases for methotrexate resistance associated with loss of RFC transport and for hereditary folate malabsorption, attributable to mutant PCFT, were determined. Future studies should continue to translate molecular insights from basic studies of RFC and PCFT biology into new therapeutic strategies for cancer and other diseases.

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“…This was determined from the recovery of radioactivity in vesicles washed by centrifugation versus filtration and from a measurement of the protein in the eluate after filtration. Concentrations for L-5-formyltetrahydrofolate are expressed as the natural (1) diastereoisomer based upon previous studies [18] from our laboratory which have shown the highaffinity carrier system in intact L1210 cell exhibited, during influx, stereospecificity at carbon 6 of L-5-formyltetrahydrofolate. The data obtained is corrected for tritium (0.1 to 0.2 pmol) substrate/mg membrane protein associating with washed vesicles after 10 sec incubation at 0 ~ which is presumed to represent surface adsorption.…”

Section: Transport Assaymentioning

confidence: 99%

Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

1984

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The effect of various anions on the mediated influx and efflux of [3H]methotrexate by L1210 cell plasma membrane vesicles in a HEPES buffer system was studied. Our results show that flux is stimulated to the same extent in either direction when SO4, Pi, or folate compounds (1,L5-CHO-folate-H4, methotrexate), but not Cl- was present in the opposite compartment. This implies the property of directional symmetry, a condition in which differential mobility of loaded and unloaded carriers occurs in both directions. We also observed a similarity in the specificity of the interaction between various anions and carrier in each orientation of the membrane, in the order, Cl- much less than Pi approximately equal to SO2-4 much less than methotrexate less than 1,L5-CHO-folate-H4. Also, the absolute differential in mobility of loaded and unloaded carrier (assumed from the extent of transstimulation obtained) varied substantially among the anions examined. No stimulation was obtained with Cl-, and stimulation was twofold with Pi, SO2-4 and methotrexate and fourfold with 1,L5-CHO-folate-H4. Transstimulation of flux from either external or internal compartment only occurred when a positive gradient of total anions was maintained in the opposite compartment. Also, no stimulation occurred when the same equivalence of two different anions are present in opposing compartments. The concentration of anions required to transstimulate [3H]methotrexate influx was increased four- to 10-fold when vesicles were equilibrated in 145 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

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Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

Cited by 71 publications

References 15 publications

Relationships between carrier-mediated transport of folate compounds by L1210 leukemia cells: Evidence for multiplicity of entry routes with different kinetic properties expressed in plasma membrane vesicles

Relationships between carrier-mediated transport of folate compounds by L1210 leukemia cells: Evidence for multiplicity of entry routes with different kinetic properties expressed in plasma membrane vesicles

The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

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