Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

Isolation and characterization of an L1210 cell line retaining the sodium-dependent carrier cif as its sole nucleoside transport activity

Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.     

The reduced folate carrier in L1210 murine leukemia cells is a 58-kDa protein

The reduced folate carrier (RFC1) is a major transporter for both natural reduced folates and antifolate chemotherapeutics. Using polyclonal antibodies targeted to epitopes at the loop between the sixth and seventh predicted transmembrane domains or the distal C-terminus, we were able to demonstrate by Western blot analysis that the molecular size of RFC1 expressed in murine leukemia L1210 cells is 58 kDa as predicted by the open reading frame of its cDNA. 46- and 38-kDa proteins detected only in plasma membrane preparations were proteolytic degradation products that appeared during membrane preparation or treatment with the conventional SDS-PAGE loading buffer. These data resolve discrepancies reported previously for the molecular size of RFC1.     

Cytotoxicity as an indicator for transport mechanism. Evidence that melphalan is transported by two leucine-preferring carrier systems in the L1210 murine leukemia cell

Melphalan, l-phenylalanine mustard, is transported by the L1210 cell through carriers of the leucine (L) type. Its initial rate of transport is inhibited by both l-leucine, a naturally occurring L system amino acid and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a synthetic amino acid which is transported by the L system in the Ehrlich ascites tumor cell. Both amino acids inhibited melphalan transport comparably in sodium-free medium. However, BCH, in medium containing sodium, was unable to reduce a component of melphalan transport which was readily inhibited by leucine but not by α-aminoisobutyric acid. Inhibition analysis indicated that leucine competes with BCH for transport but that a portion of leucine transport is not readily inhibited by BCH. These results suggest that in the L1210 cell melphalan is transported equally by a BCH-sensitive, sodium-independent L system and a BCH-insensitive, sodium-dependent L system.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Evidence for a membrane sulfhydryl associated with resistance to melphalan in a murine L1210 leukemia line

Murine L1210 leukemia cells, which display sensitivity or resistance to the chemotherapeutic alkylating agent, melphalan, are equivalently sensitive or resistant to the poorly permeable mercurial, p-chloromercuribenzenesulfonate. Cells of both lines do not differ in sensitivity to the sulfhydryl reagents, N-ethylamaleimide, iodoacetamide and iodoacetate or to the glutathione transferase substrates 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride. The results are interpreted in terms of the known biological reactivity of p-chloromercuribenzenesulfonate as a selective reagent for sulfhydryl groups of membrane proteins associated with monovalent cation permeability.     

Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: involvement of a membrane receptor and a cytosolic protein

A radioiodinated photoaffinity analogue of methotrexate, N alpha-(4-amino-4-deoxy-10-methyl-pteroyl)-N epsilon-(4-azidosalicylyl)-L- lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells [Price, E. M., & Freisheim, J. H. (1987) Biochemistry 26, 4757-4763]. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degrees C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degrees C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxicity and induction of apoptosis by 4-amino-3-acetylquinoline in murine leukemia cell line L1210

Nitrogen heterocyclic compounds are used in the pharmaceutical industry, in medicine and in agriculture for their biological activity. 4-Amino-3-acetylquinoline, a new synthetically prepared quinoline derivative, was the most effective compound in our primary cytotoxic screening. In this study, we evaluated cytotoxic/antiproliferative activity of quinoline using murine leukemia cell line L1210. Its ability to induce apoptosis was studied, too. Quinoline derivative acted cytotoxically on tumor cell line L1210, the IC(100) value were 50 microg/ml (for 24 h), 25 microg/ml (for 48 h) and 10 microg/ml (for 72 h). The IC(50) values was found to be less than 4 microg/ml, a limit put forward by the National Cancer Institute (NCI) for classification of he compound as a potential anticancer drug. The cytotoxic concentrations of 4-amino-3-acetyl quinoline induced morphological changes of L1210 cells and the apoptotic DNA fragmentation.     

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.     

Vinca alkaloid-induced tubulin spiral formation correlates with cytotoxicity in the leukemic L1210 cell line

The ability of a class of C-20' modified vinca alkaloid congeners to induce tubulin spiral formation was investigated relative to their ability to inhibit microtubule assembly, their cytotoxicity against a leukemic cell line, L1210, and their measured and calculated partition coefficients. These studies were prompted by the observation that the energetics of vinca alkaloid-induced tubulin spiral polymers, or spiraling potential, is inversely related to their clinical dosage and are aimed at the long-term goal of developing the ability to predict the cytotoxic and antineoplastic properties of antimitotic drugs. We demonstrate here that vinca-induced tubulin-spiraling potential is significantly correlated with cytotoxicity against L1210 cells. This is consistent with the size of spirals formed being proportional to the relaxation time for polymer redistribution, the lifetime of cell retention, and effects on microtubule ends and dynamics. Spiraling potential also correlates with calculated but not measured partition coefficients. Surprisingly, spiraling potential does not correlate with the ability to inhibit microtubule formation with purified tubulin or microtubule protein. For the set of C-20' modified compounds studied, the largest inhibitory effects on spiraling potential and cytotoxicity are caused by multiple sites of halogen (-F, -Cl) substitution with the introduction of increased rigidity in the ring. This suggests the C-20' position interacts with a hydrogen bond acceptor or an electrophilic region on the protein that electrostatically disfavors halogen substitutions. These studies are discussed in terms of the cellular mode of action of antimitotic drugs, particularly the importance of microtubule dynamics during mitosis and the factors that regulate those dynamics.     

Isolation and characterization of a mutant of L1210 murine leukemia deficient in nitrobenzylthioinosine-insensitive nucleoside transport

L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.     

Manipulating cofactor binding thermodynamics in an artificial oxygen transport protein

We report the mutational analysis of an artificial oxygen transport protein, HP7, which operates via a mechanism akin to that of human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of 13. Paradoxically, it also decreases heme binding affinity by a factor of 5 in the reduced state and 60 in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates for that. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins.     

An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.     

A nonfunctional protein in human leukemia cells reacts with antiserum to dihydrofolate reductase from L1210 leukemia cells

Antiserum raised in chickens to dihydrofolate reductase purified from L1210 leukemia cells by affinity chromatography inhibited the catalytic activity and the binding of methotrexate by the enzyme. Lysates of human chronic myelogenous leukemia cells, which had neither catalytic activity for dihydrofolate reductase nor binding of methotrexate, blocked the inhibiting effect of the antiserum on the function of the enzyme in L1210 cell lysates. In double immunodiffusion, these human leukemia cell lysates formed a single precipitin line against the antiserum. These findings indicate that nonfunctional dihydrofolate reductase in human leukemia cells share an antigenic determinant(s) with a functional form of the enzyme from L1210 murine leukemia cells.     

Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.