Enhancement of Folate Analogue Transport Inward in LI 210 Cells during Methotrexate Therapy of Leukemic Mice: Evidence of the Nature of the Effect, Possible Host Mediation, and Pharmacokinetic Significance'

Studies are described that sought the basis for a discrepancy in values for a key kinetic parameter of methotrexate transport (influx Vn,»,( in LI 210 cells derived alternately from biochemical or pharmacokinetic measurements. Our results show that, within a short period of time following administration of a therapeutic dose of methotrexate to leukemic mice, influx of this folate analogue measured in 1,121(1 cells removed from these mice was markedly stimulated. Enhancement of |3H)methotrexate influx in these cells was observed within 15 min of drug administration, was maximum (up to 3-fold) within 2 to 3 h, then decreased with time until 24 h when influx was at the control level. Measurements of [3H]methotrexate influx in cells removed from drugtreated mice were made after a period of incubation in drug-free medium to allow for efflux of exchangeable drug. Enhanced influx of [3H]methotrexate was accounted for by an increase in influx Vm.x(influx K„ was unchanged) and was further enhanced (to a total of 5-fold) by coadministration of leucovorin. Also, enhancement of influx of [3H]methotrexate in 1,1210 cells did not occur following administration of l-/î-i>-arabinofuranosylcytidine at a therapeutically equivalent dose to leukemic mice or following exposure of these cells to methotrexate or methotrexate with leucovorin during growth in culture. Methotrexate therapy did not affect all transport systems since the same therapy of leukemic mice had no effect on influx of the purine nucleoside analogue, 9-/J-D-arabinofuranosyl-2-fluoroadenine, in these same I 1210 cells. These findings suggest that stimulation of [3H]methotrexate influx in LI 210 cells during therapy with this folate analogue was not due to transstimulation during exchange between folate compounds and was not related to the antiproliferative effect of methotrexate on these tumor cells. The coadministration of cycloheximide with methotrexate to leukemic mice at a dose which markedly inhibited 3H-leucine incorporation into LI 210 cell protein severely diminished the stimulation of [3H]methotrexate influx. However, in LI 210 cells removed from leukemic mice treated with methotrexate, there was no increase compared to control cells in affinity labeling with the yV-hydroxysuccinimide ester of (3H|methotrexate. This suggested that the effect of cycloheximide was not on increased synthesis of folate transporter and that increased rate of translocation of folate transporter, rather than increased amount of transporter, accounted for the increase in (3H]methotrexate influx. Although the stimulation of [3H]methotrexate influx observed in 1,12111 cells removed from mice pretreated with this antifolate did not seem to result from the increased content of folate transporter, the nullification of this effect by cycloheximide does suggest that there is a requirement for new protein synthesis elsewhere. Since the stimulation of [3H|methotrexate influx could not be demonstrated by preexposure of growing LI 210 cells in culture to methotrexate with or without leucovorin, a role for the host is indicated in the mediation of this effect. Pharmacokinetic implications of these results are discussed. INTRODUCTION Notions as to the pharmacological behavior of various antineoplastic agents in animal tumor models or in patients, par ticularly, in proliferative tissue compartments, have often relied upon (see Refs. 1 to 4 pertaining to antifolates) measurements of specific pharmacokinetic parameters in isolated cell systems in vitro. However, to what extent such measurements have significant predictive value has rarely been addressed. Our earlier studies of folate analogue transport in L1210 cells have, in fact, revealed marked discrepancies in values obtained for certain kinetic parameters for transport when derived either in vitro (reviewed in Ref. 4) or from pharmacokinetic measure ments in vivo (5). Specifically, the value for Vmaxof methotrex ate influx in these tumor cells when derived from the latter studies was substantially greater than values obtained by direct measurement of this kinetic parameter in vitro. Pharmacoki netic parameters for methotrexate and related folate analogues have useful applications (6-10) in the development of new, more efficacious analogues or improved clinical protocols. For this reason, we have examined the basis for the observed dis crepancy in this parameter for methotrexate influx in these tumor cells. Surprisingly, our results show that, within a short period of time following administration of a therapeutic dose of methotrexate to LI 210 leukemic mice, the transport of this folate analogue inward (influx) in LI210 cells was appreciably increased. This enhancement of influx (up to 3-fold) was quan titatively accounted for by an increase in influx Vmax. Also, further enhancement of [3H]methotrexate influx (an additional 2to 3-fold) occurred when a molar equivalent dose of leuco vorin was coadministered with methotrexate. In contrast, the same effect was not obtained on another system mediating the influx of [3H]F-Ara A" in these LI210 cells. Other studies showed that the stimulation of [3H]methotrexate influx did not occur following administration of a therapeutically equivalent dose of Ara C, thus suggesting that the stimulation seen with methotrexate was unrelated to the antiproliferative effect of this antifolate. The stimulation in methotrexate influx seen following methotrexate administration might be host mediated, since no effect of pretreatment could be demonstrated in LI 210 cells growing in culture. We also found that the stimulation of influx did not involve an increase in the amount of an affinitylabeled surface protein but could be abrogated by the coadmin istration of cycloheximide. Received 3/26/87; revised 6/29/87; accepted 7/23/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by Grants CA 08748, CA 18856, and CA 22764 from the National Cancer Institute; CH-26 from the American Cancer Society; and the Else I '. Pardee Foundation. 1To whom requests for reprints should be addressed, at Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. 3 Present address: A. H. Robbins Corp., 1211 Sherwood Ave., Richmond. VA 23220. MATERIALS AND METHODS General. Harvesting of tumor cells from the peritoneal cavity and maintenance of L1210/V cells in (female C57BL/6 x DBA/2 F,) (hereafter called B6D2F|) mice (purchased from the National Cancer Institute) have been described (4). All folate compounds were prepared 'The abbreviations used are: F-Ara-A, 9-/3-o-arabinofuranosyl-2-fluoroadenine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate; NHS, JV-hydroxysuccinimide; EDC, l-ethyI-3-<3-dimethylaminopropyl)carbodiimide; Ara C, l-ßD-arabinofuranosylcytidine; V,¡fjs. kinetic parameter of maximum influx at 37*C; Ai7, efflux rate constant at 37"C; TCA, trichloroacetic acid.