I.David Goldman

Rapid separation of low molecular weight solutes from liposomes without dilution.

Abstract Liposomes can be separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1- or 5-ml plastic syringes. Excess fluid is first removed from the Sephadex beads by centrifugation and a mixture of liposomal entrapped and free solute is applied to the column bed. The centrifugation is repeated forcing the liposomal material through the column into a test tube while the free solute is quantitatively retained in the Sephadex. The procedure is applicable to a variety of solutes and 92 to 100% recovery is achieved for both charged and neutral liposomes. This technique has advantages over other methods for separating extraliposomal solutes from liposomes. Numerous samples can be processed simultaneously within minutes with no dilution of the liposomal preparation. Nonentrapped solute within the Sephadex can be easily recovered in a small volume of water or buffer.

The cellular pharmacology of methotrexate

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Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells.

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibiting of [3H]folic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6R) diasterceoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

A method for the synchronization of cultured cells with aphidicolin: Application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase

The DNA polymerase alpha inhibitor, aphidicolin, was employed to synchronize large-scale suspension cultures (10(9) cells) of murine L1210 leukemia cells. On the basis of the doubling time and cell cycle distribution for logarithmically growing L1210 cells, a synchronization protocol was devised involving a temporal sequence of two 12-h exposures to aphidicolin, separated by an 6-h interval in drug-free medium. After the second aphidicolin treatment, resuspension of cells into drug-free medium resulted in the rapid onset of DNA synthesis as assessed by [3H]thymidine incorporation and DNA fluorescence with flow cytometry. By 6 h after aphidicolin removal, the cells progressed into the G2-M phase and cell division was initiated. DNA synthesis was minimal during this time and remained low through 9 h when the majority of the cells were in G1 phase. Only low levels of cytotoxicity were observed when L1210 cells were treated with aphidicolin in this fashion. The levels of both thymidylate synthase and dihydrofolate reductase were relatively constant during cell cycle transit, following release from the aphidicolin blockade. Similarly, the levels of the corresponding mRNA transcripts for these enzymes, measured by Northern blot hybridizations, remained essentially unchanged through most of the cell cycle, increasing approximately twofold only as the cells entered G1 phase. Whereas intracellular dihydrofolate reductase catalytic activity was relatively unchanged throughout the cell cycle, as reflected in the metabolism of [3H]folic acid to reduced folate forms, a marked increase in in situ thymidylate synthase activity occurred during S phase that was tightly linked to the rate of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells

This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-beta (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-beta and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 microM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-beta exhibited an influx kappa t one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO4(2-) was inhibitory. Anions enhanced MTX binding to folate receptor-beta with isethionate > SO4(2-) > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-beta-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.

Hydroxylation of 4-amino-antifolates by partially purified aldehyde oxidase from rabbit liver.

This paper explores the interaction between 4-amino-antifolates and aldehyde oxidase (aldehyde: O2 oxidoreductase, EC 1.2.3.1) that was purified 60- to 120-fold from rabbit liver with yields of 5-15%. The purification procedure consisted of one heat and two ammonium sulfate precipitations followed by chromatography on hydroxylapatite and then Sephacryl S-200. Analysis of initial rates of hydroxylation of methotrexate, aminopterin and dichloromethotrexate indicated an order of affinities of dichloromethotrexate (10 microM) greater than methotrexate (35 microM) greater than aminopterin (272 microM). There was no difference in the Vmax of methotrexate and dichloromethotrexate (248 and 231 nmoles/min/mg protein respectively); aminopterin (130 nmoles/min/mg protein) was less than that of the other two. The Vmax/Km ratios were 24.1, 7.20 and 0.48 for dichloromethotrexate, methotrexate and aminopterin respectively. This enzyme preparation also mediated the hydroxylation of methotrexate polyglutamyl derivatives with a decrease in the rates of hydroxylation, as the total number of glutamyl residues was increased to four, a consequence of a marked increase in Km values and/or decrease in Vmax; the ratios of the Vmax/Km for the di-, tri-, and tetraglutamates were 0.94, 0.31 and 0.21 respectively. This low activity of the polyglutamyl derivatives of methotrexate for aldehyde oxidase is consistent with the observations that the predominant forms of 4-amino-antifolate polyglutamates found in human liver after administration of methotrexate are the polyglutamyl derivatives of the parent compound. Finally, substrate inhibition for methotrexate and dichloromethotrexate was observed at concentrations in excess of 150 and 30 microM, respectively, about 5- and 3-fold higher than their respective Km values. Hence, while dichloromethotrexate had the lowest Km for aldehyde oxidase amongst the 4-amino-antifolates studied, the actual rates of hydroxylation depended upon the concentration employed because of substrate inhibition. Aminopterin was a very poor substrate for this enzyme at low and saturating concentrations. These properties of the hydroxylation of 4-amino-antifolates may be of importance in the design of clinical regimens with these agents--in particular, regimens that employ infusion of these drugs into the hepatic artery. However, the relevance of these observations to the hydroxylation of 4-amino-antifolates by human liver remains to be established.

Comparison of methotrexate polyglutamylation in l1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor: Lack of evidence for influx route-specific effects

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Compartmentation of intracellular folates: Failure to interconvert tetrahydrofolate cofactors to dihydrofolate in mitochondria of L1210 leukemia cells treated with trimetrexate

Following exposure of L1210 leukemia cells to antifolates, tetrahydrofolate-dependent purine and pyrimidine biosyntheses are blocked despite the presence of the major portion of tetrahydrofolate cofactors. Previous studies from this laboratory demonstrated that this cannot be due to direct inhibition of thymidylate synthase by dihydrofolate polyglutamates or other endogenous folates and suggested that this phenomenon is due to compartmentation of tetrahydrofolate cofactors unavailable for interconversion and/or oxidation when dihydrofolate reductase activity is abolished by antifolates. The present paper evaluates the possibility that tetrahydrofolate cofactors in subcellular organelles, in particular, mitochondria, are unavailable for oxidation by thymidylate synthase. Particulate and cytosolic fractions were obtained from L1210 cells following homogenization and differential centrifugation. The crude mitochondrial fraction contained 20.1% of the total folate pool and included 5-formyltetrahydrofolate, 10-formyltetrahydrofolate and tetrahydrofolate in proportions similar to intact cells. The cytosolic fraction had an increased proportion of tetrahydrofolate and decreased proportions of 5-formyl- and 10-formyltetrahydrofolate relative to intact cells or the particulate fraction. Exposure of cells to 10 microM trimetrexate for 30 min produced approximately 45% interconversion of tetrahydrofolate cofactors to dihydrofolate in the cytosolic fraction, a level much greater than that observed in whole cell extracts (25-30%), but had no effect on folate pools in the crude mitochondrial fraction. These data indicate that subcellular compartmentation accounts, in part, for the failure to oxidize tetrahydrofolate cofactors to dihydrofolate in the presence of antifolate levels that abolish dihydrofolate reductase activity.

Membrane Transport of Antifolates as a Critical Determinant of Drug Cytotoxicity

There are few areas in therapeutics in which membrane transport of pharmacologic agents has been subjected to as intense study as in the treatment of malignant diseases with cytotoxic agents. Unlike the antibacterial or antifungal agents, which may selectively inhibit biochemical processes unique to the bacterial or fungal organism, the agents which comprise the current armamentarium of the cancer chemotherapist produce toxic effects on both tumor and susceptible host tissues. The lack of understanding of the basic biochemical differences between normal and malignant cells require the utilization of subtle techniques, often empirical, to achieve “selective” effects of the agents in an attempt to minimize toxicity to susceptible host tissues while maximizing toxicity to the tumor. The better understood of these techniques usually exploit quantitative rather than qualitative differences in the tumor vs. host cell rates of (a) cellular proliferation, (b) drug activation or inactivation, (c) drug transport across the cell membrane, and (d) drug interaction with target sites within the cell.

Efflux in isolated hepatocytes as a possible correlate of secretion in, vivo: Induced exit of the folic acid analog methotrexate, by dibutyryl cyclic AMP or isobutyl methyl xanthine

Abstract Dibutyryl cyclic AMP and isobutyl methyl xanthine induce release of freely exchangeable methotrexate as well as a small component of apparently bound drug from freshly isolated rat hepatocytes; methotrexate polyglutamate derivatives are retained. These observations, as well as the energy dependence of methotrexate efflux induced by dibutyryl cyclic AMP suggests that this may represent the induction of a “secretory” phenomenon in which drug is released into the capillary sinusoid and/or the bile canaliculus when the hepatocyte is in its normal spatial orientation in the liver lobule in , vivo . Because there is evidence that this folic acid analog and bile salts utilize the same transport mechanism in these cells, this phenomenon may have general physiological as well as pharmacologic relevance and the isolated hepatocyte may be a useful model system to study mechanisms of hepatic secretion at the cellular level.