ATB0,+ [SLC6A14 (solute carrier family 6 member 14)] is an Na+/Cl−-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB0,+ in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB0,+. The transport process is Na+/Cl−-dependent with an Na+/Cl−/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of α-methyltryptophan as a blocker, not a transportable substrate, for ATB0,+. ATB0,+ can transport 18 of the 20 proteinogenic amino acids. α-Methyltryptophan blocks the transport function of ATB0,+ with an IC50 value of ∼250 μM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that α-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB0,+ is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB0,+-positive tumour cells with α-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB0,+-negative cell lines are not affected. The blockade of ATB0,+ in these cells with α-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB0,+ as a drug target for cancer chemotherapy.
The cytosolic enzyme IDO (indoleamine 2,3-dioxygenase) is an important component of tumour-associated immune tolerance [1–4]. The relevance of IDO to immune tolerance was first demonstrated by Munn et al. , showing that inhibition of the enzyme leads to rejection of allogeneic fetuses by disrupting the tolerance of the maternal immune system during pregnancy. Tumours evade the surveillance of the immune system by inducing IDO in tumour cells themselves, stromal cells or immune cells present in the tumour-draining lymph nodes [6–9]. It is believed that tumour-associated up-regulation of IDO leads to enhanced breakdown of the essential amino acid tryptophan from the surroundings, with the resultant tryptophan depletion leading to prevention of T-cell activation [1–4]. Thus induction of IDO is a critical determinant of tumoral immune tolerance. Therefore inhibition of IDO is expected to reverse this process, causing activation of the immune system and killing of tumour cells. 1-Methyltryptophan is widely used as a pharmacological inhibitor of IDO  in mouse models, and is currently in preclinical development phase for the treatment of cancer [1–4]. To produce its therapeutic effect, 1-methyltryptophan has to enter the IDO-expressing cells first, but very little is known about the identity of the plasma membrane transporters that are responsible for this entry process.
ATB0,+ is an amino acid transporter with special functional features . It transports 18 of the 20 proteinogenic amino acids, and its transport process is energized by three different driving forces, namely an Na+ gradient, a Cl− gradient and membrane potential. Theoretically, this transporter has the ability to concentrate its substrates inside the cells more than 1000-fold. ATB0,+ has been cloned from human and rodent tissues, and the function of the cloned transporter has been characterized in heterologous expression systems [12–14]. According to the HUGO (Human Genome Organisation) nomenclature, ATB0,+ is identified as SLC6A14 (solute carrier family 6 member 14; i.e. the 14th member of the solute carrier gene family SLC6). Functional expression in Xenopus laevis oocytes has demonstrated that the transport process mediated by ATB0,+ is electrogenic, associated with the transfer of net positive charge into the oocytes [12,13]. Recent studies in our laboratory have unravelled the therapeutic potential of this transporter owing to its ability to transport a variety of pharmacological agents, including NOS (nitric oxide synthase) inhibitors  and amino acid-based prodrugs of the antiviral agents acyclovir and ganciclovir [16,17]. There is emerging evidence for tumour-associated up-regulation of ATB0,+ [18,19]. The expression of this transporter is markedly induced in colorectal cancer  and cervical cancer , and the up-regulation is demonstrable at the mRNA level as well as at the protein level. Since ATB0,+ can transport almost all amino acids, the only exceptions being glutamate and aspartate, tumour cells can meet their increasing demand for amino acids simply by turning on a single gene (SLC6A14) coding for the transporter. It is therefore likely that the up-regulation of ATB0,+ is not restricted to colorectal and cervical cancers. Even though the expression of this transporter has not been investigated in cancers of other tissues, we hypothesize that overexpression of this transporter may be a widespread phenomenon in cancer. The potential relevance of the transport function of ATB0,+ to tumour growth is the basis of this hypothesis. Since the basal expression of the transporter in different tissues is low under normal conditions, the tumour-associated up-regulation of the transporter could be exploited for tumour-specific delivery of anticancer drugs, leaving the adjacent normal cells unaffected by the effects of such drugs. The ability of the transporter to recognize structurally diverse pharmacological agents lends credence to its therapeutic potential in cancer treatment.
ATB0,+ accepts tryptophan as a substrate with high affinity [12,13]. Therefore we hypothesized that the transporter may participate in the cellular uptake of 1-methyltryptophan. Since the transporter is up-regulated markedly in cancer, active cellular uptake of 1-methyltryptophan via the transporter may facilitate the inhibition of tumour-associated IDO in a specific manner. With this rationale, the present investigation was initiated to study the transport of 1-methyltryptophan and other tryptophan derivatives via ATB0,+. These studies have not only shown that the IDO inhibitor 1-methyltryptophan is indeed a transportable substrate for ATB0,+, but also identified α-methyltryptophan as a blocker of the transporter. Since the transporter is up-regulated in tumour cells, blocking of the transporter can be expected, in theory, to starve the tumour cells of amino acids, thus causing growth arrest. We tested this in breast cancer cell lines. These studies have shown that treatment of cancer cell lines overexpressing ATB0,+ with α-methyltryptophan does induce growth arrest, suggesting that the transporter can be exploited as a potential drug target for cancer chemotherapy with identification of high-affinity blockers of the transporter.
MATERIALS AND METHODS
[3H]Glycine (specific radioactivity=30 Ci/mmol), D-[3H]serine (specific radioactivity=20 Ci/mmol) and L-[14C]valine (specific radioactivity=260 mCi/mmol) were purchased from Moravek Biochemicals (Brea, CA, U.S.A.). Tryptophan and its derivatives, including 1-methyl-DL-tryptophan, 1-methyl-L-tryptophan, 1-methyl-D-tryptophan and α-methyl-DL-tryptophan, were from Sigma. The numbers in tryptophan derivatives refer to the position of the substitutions (methyl or hydroxy group) in the indole ring, with nitrogen in the ring being position 1 and the carbon atoms in the ring being counted counter-clockwise. α-Methyltryptophan has the methyl group substitution at the α-carbon of the aliphatic side chain. N-Acetyltryptophan has the acetyl group substitution at the α-amino group in the side chain. The mouse ATB0,+ cDNA was cloned from a colon cDNA library and its transport function has been established in previous studies . The human ATB0,+ cDNA was cloned from an MCF7 cell (a human mammary epithelial cancer cell line) cDNA library and the functional identity of the clone has been established by its ability to mediate Na+- and Cl−-coupled transport of glycine and D-serine in a heterologous expression system using mammalian cells (T. Hatanaka and V. Ganapathy, unpublished work). Human LAT1 cDNA was cloned from a human placental cell line and its functional identity has been established previously in a mammalian cell heterologous expression system with co-expression of 4F2hc .
Functional expression of cloned ATB0,+ and LAT1/4F2hc in HRPE cells (human retinal pigment epithelial cells)
Mouse and human ATB0,+ and human LAT1/4F2hc cDNAs were expressed functionally in an HRPE cell line using the vaccinia virus expression technique as described previously . This procedure involves infection of the cells with a recombinant vaccinia virus carrying the gene for T7 RNA polymerase, followed by Lipofectin®-mediated transfection of the cells with plasmid DNA in which the cDNA insert is under the control of T7 promoter. Glycine was used as the substrate for ATB0,+. Transport of 10 μM glycine (radiolabelled glycine, 0.05 μM; unlabelled glycine, 9.95 μM) in cDNA-transfected cells was measured at 37 °C for 30 min, representing linear uptake rates. The transport buffer was 25 mM Hepes/Tris (pH 7.5) containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4 and 5 mM glucose. Transport was terminated by aspiration of the transport buffer followed by three washes with 2 ml of ice-cold transport buffer. The cells were lysed with 0.5 ml of 1% SDS in 0.2 M NaOH and the lysate was used for determination of radioactivity. Interaction of tryptophan and its derivatives with the transporter was assessed by monitoring the ability of these compounds to inhibit ATB0,+-mediated glycine transport. In initial experiments, we compared glycine uptake between cells transfected with vector alone and cells transfected with ATB0,+ cDNA to assess the relative levels of constitutive glycine uptake rate. These experiments revealed that glycine uptake in vector-transfected cells was less than 3% of glycine uptake in cDNA-transfected cells. Therefore all subsequent studies were done only with cDNA-transfected cells, considering the constitutively expressed glycine uptake as negligible.
L-Valine was used as the substrate for LAT1/4F2hc. Transport of L-[14C]valine (0.75 μM) was measured in vector-transfected cells and in cDNA-transfected cells in parallel in the same buffer described above, but with a 5 min incubation instead of a 30 min incubation. Parallel uptake measurements in vector-transfected cells and cDNA-transfected cells were necessary because of a significant constitutive expression of system L activity in these cells . Co-expression of human LAT1 cDNA and human 4F2hc cDNA in these cells increased the activity for valine transport by 3-fold, showing that the transport activity in vector-transfected cells was ∼30% of the transport activity in cDNA-transfected cells. The interaction of tryptophan derivatives with LAT1/4F2hc was evaluated by assessing the effects of these compounds (1 mM) on the transport of valine in vector-transfected cells and in cDNA-transfected cells and then calculating the cDNA-specific transport activity in the presence and absence of these derivatives.
Functional expression of ATB0,+ in X. laevis oocytes
Capped cRNA from the cloned mouse or human ATB0,+ cDNA was synthesized using the mMESSAGE mMACHINE kit (Ambion, Austin, TX, U.S.A.). Mature oocytes (stage IV or V) from X. laevis were injected with 50 ng of cRNA. Uninjected oocytes served as controls. The oocytes were used for electrophysiological studies 3–6 days after cRNA injection. Electrophysiological studies were performed by the two-microelectrode voltage-clamp method . Oocytes were superfused with an NaCl-containing buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 3 mM Hepes, 3 mM Mes and 3 mM Tris, pH 7.5), followed by the same buffer containing tryptophan derivatives. The membrane potential was clamped at −50 mV. The differences between the steady-state currents measured in the presence and absence of substrates were considered as the substrate-induced currents. In the analysis of the saturation kinetics of substrate-induced currents, the kinetic parameter K0.5 (i.e. substrate concentration necessary for induction of half-maximal current) was calculated by fitting the values of the substrate-induced currents to the Michaelis–Menten equation. The Na+ and Cl− activation kinetics of substrate-induced currents were analysed by measuring the substrate-specific currents in the presence of increasing concentrations of Na+ (concentration of Cl− kept constant at 100 mM) or in the presence of increasing concentrations of Cl− (concentration of Na+ kept constant at 100 mM). In these experiments, the composition of the superfusion buffer was modified to contain 2 mM potassium gluconate, 1 mM MgSO4 and 1 mM calcium gluconate in place of KCl, MgCl2 and CaCl2 respectively. The data from these experiments were analysed by the Hill equation to determine the Hill coefficient (h; number of Na+ and Cl− ions involved in the activation process). The kinetic parameters were determined by using the commercially available computer program Sigma Plot, version 6.0 (SPSS, Chicago, IL, U.S.A.). Uninjected oocytes were used to determine the currents induced by tryptophan derivatives due to entry via transporters that may be constitutively expressed in the oocytes. At a concentration of 2.5 mM, the currents induced by these compounds were in the range of 0–3 nA in uninjected oocytes. These currents were less than 2% of the corresponding currents induced in ATB0,+ cRNA-injected oocytes. Therefore the endogenous currents associated with the constitutively expressed transporters were not considered in the analysis of kinetic parameters of ATB0,+ transport activity.
Mammary epithelial cell lines and their culture
MCF7, T-47D, ZR-75.1, MB-231, MB-361 and MB-453 cell lines were purchased from the A.T.C.C. (Manassas, VA, U.S.A.) and cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 1 mM pyruvate. HMEC (human mammary epithelial cell) (Clonetics, Walkersville, MD, U.S.A.) and MCF10A (A.T.C.C.) cell lines were cultured in Complete Mammary Epithelial Growth medium (Cambrex, Walkersville, MD, U.S.A.) containing 100 ng/ml cholera toxin and 2 mM pyruvate. HBL100 cell line (originally obtained from Dr Sukumar, Johns Hopkins University, Baltimore, MD, U.S.A.) was cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 1 mM pyruvate. Among the nine cell lines examined in the present study, HMEC, HBL100 and MCF10A are non-malignant because these cell lines do not form a tumour when xenografted into nude mice. In contrast, the other remaining six cell lines (MCF7, T-47D, ZR-75.1, MDA-MB231, MDA-MB361 and MDA-MB453) are malignant based on their ability to form tumour when xenografted in nude mice.
Uptake measurements in cell lines
Cells were seeded and grown in 24-well culture plates to confluency and used for uptake measurements. Radiolabelled glycine or D-serine was used as the substrate to determine the transport function of ATB0,+. Uptake measurements were routinely made in an NaCl-containing buffer (25 mM Hepes/Tris buffer, pH 7.4, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4 and 5 mM glucose). Ion dependence of the transport process was evaluated by studying the uptake activity in buffers of varying compositions. NaCl in the uptake buffer was replaced iso-osmotically with NMDG (N-methyl-D-glucamine) chloride to prepare an Na+-free buffer, and with sodium gluconate to prepare a Cl−-free buffer. Linear uptake rates were determined by employing a 10 min incubation period, based on time course experiments. Na+-activation kinetics was analysed by studying the uptake rates in the presence of varying concentrations of Na+ (2.5–140 mM) in the uptake buffer with Cl− concentration kept constant at 140 mM. This was done by replacing NaCl with appropriate concentrations of NMDG. Cl−-activation kinetics was analysed by studying the uptake rates in the presence of varying concentrations of Cl− (2.5–100 mM) in the uptake buffer with Na+ concentration kept constant at 140 mM. This was done by replacing NaCl with appropriate concentrations of sodium gluconate. Uptake data for Na+- and Cl−-activation kinetics were analysed using the Hill equation, and values for K0.5 for Na+ and Cl− and for the Hill coefficient (h) were determined by linear and non-linear regression methods. A commercially available software program (Sigma Plot, version 6.0; SPSS) was used for this purpose. Substrate specificity of the uptake process was evaluated by monitoring the effects of unlabelled amino acids (5 mM) on the uptake of radiolabelled glycine or D-serine (5 μM: radiolabelled substrate, 0.05 μM; unlabelled substrate, 4.95 μM).
The kinetic nature of inhibition of D-serine uptake by α-methyl-DL-tryptophan was investigated in MCF7 cells by monitoring the uptake at increasing concentrations of D-serine (range, 50–2000 μM) in the absence and presence of 100 μM α-methyl-DL-tryptophan. The kinetic parameters were calculated by linear regression (Eadie–Hofstee plots) and the values were confirmed by non-linear regression.
Analysis of ATB0,+ expression
For the analysis of the expression of ATB0,+ mRNA, RNA prepared from the cell lines was used. RNA (2 μg) was reverse-transcribed using the GeneAmp® PCR system (Roche). HPRT1 (hypoxanthine–guanine phosphoribosyltransferase 1) mRNA was used as an internal control. Human ATB0,+-specific PCR primers were designed based on the nucleotide sequences available in GenBank® Nucleotide Sequence Database (upstream primer: 5′-GAAGGAGAAAGTGTCGGCTTCA-3′; downstream primer: 5′-TACCACCTTGCCAGACGATTTG-3′). For the analysis of ATB0,+ protein, Western blot was used. Cell lysates were prepared by sonication in 10 mM Tris/HCl buffer (pH 7.6) containing protease inhibitors (50 mM NaF, 0.2 mM vanadate, 1 mM PMSF, 5 μg/ml aprotinin, 1 μg/ml pepstatin A and 2 μg/ml leupeptin) and 1% Triton X-100. Protein (50 μg) was fractionated by SDS/PAGE gels and transferred on to a Protran nitrocellulose membrane (Schleicher and Schull). Membranes were blocked with 5% (w/v) non-fat dried skimmed milk powder, exposed to primary antibody at 4 °C overnight followed by treatment with an appropriate secondary antibody, conjugated to horseradish peroxidase, at room temperature for 1 h, and developed by SuperSignal Western-blotting system (Pierce). The details of the primary antibody have been published previously [18,19].
Expression of IDO, LAT1, GLYT1 and GLYT2
The expression of IDO, LAT1, GLYT1 and GLYT2 in the breast epithelial cell lines was examined by RT–PCR (reverse transcription–PCR). There are two isoforms of IDO in humans, namely IDO1 and IDO2. In addition, there is another related enzyme, known as TDO (tryptophan-2,3-dioxygenase), which also mediates the breakdown of tryptophan. We investigated the expression of IDO1, IDO2 and TDO by RT–PCR in nine different HMEC cell lines (three non-malignant cell lines and six malignant cell lines). The primer pairs used were as follows: 5′-GCAAATGCAAGAACGGGACAC-3′ (upstream) and 5′-TCAGGGAGACCAGAGCTTTCACAC-3′ (downstream) for IDO1, 5′-ACAGGACCACTTGCTGACAGCTTA-3′ (upstream) and 5′-ACGTGGGTGAAGGATTGACTCCAA-3′ (downstream) for IDO2, and 5′-AAACCTCCGTGCTTCTCAGACAGT-3′ (upstream) and 5′-TGAAGTCCAAGGCTGTCATCGTCT-3′ (downstream) for TDO. The primer pairs for the various amino acid transporters were as follows: 5′-GTGTGATGACGCTGCTCTACG-3′ (upstream) and 5′-GATGATGGTGAAGCCGATGC-3′ (downstream) for human LAT1, 5′-CAACTGTGCCACCAGCGTCTA-3′ (upstream) and 5′-ACGCCCAAGGTCACATAGGTCT-3′ (downstream) for human GLYT1, and 5′-GACGAGTTTCCCAAGTACCTAC-3′ (upstream) and 5′-CAGTTAGGATAGCGGTAAGAGC-3′ (downstream) for human GLYT2. HPRT was used as the internal control.
Colony formation assay
The colony formation assay was performed as described previously . Mammary epithelial cells were seeded on to 6-well plates (10000 cells per well) in DMEM. After 24 h, cells were exposed to varying concentrations of α-methyl-DL-tryptophan or 1-methyl-DL-tryptophan for 2 weeks, changing medium every 2 days. Cells were washed with PBS and fixed in 100% (v/v) methanol for 30 min followed by staining with KaryoMAX Giemsa stain for 1 h. The wells were washed with water and dried overnight at room temperature. Finally, the dye in the wells was dissolved with 1% SDS in 0.2 M NaOH for 1 h, and the attenuance of the released dye was measured at 630 nm.
Cell cycle analysis
MCF10A (an ATB0,+-negative non-malignant cell line), MCF7 (an ATB0,+-positive malignant cell line) and MDA-MB453 (an ATB0,+-negative malignant cell line) cells were cultured in 6-well plates in a regular culture medium in the presence or absence of α-methyl-DL-tryptophan (2.5 mM) for 24, 48 and 72 h. Cells were fixed in 50% (v/v) ethanol, treated with 0.1% sodium citrate, 1 mg/ml RNase A and 50 μg/ml propidium iodide, and subjected to FACS (Becton Dickinson FACSCalibur™) analysis. The amount of DNA in these cells, detected as chromosome content by propidium iodide binding, was used to identify cells in various stages of cell cycle: G1/G0, 2N; S, 2–4 N; G2/M, 4N (where N is the normal chromosome content).
Interaction of tryptophan derivatives with cloned ATB0,+ expressed heterologously in HRPE cells
We first studied the interaction of 1-methyltryptophan and other derivatives of tryptophan with the cloned mouse ATB0,+ by functionally expressing the clone in HRPE cells. HRPE cells exhibit very low basal glycine uptake activity, and the uptake increases more than 30-fold when mouse ATB0,+ cDNA is expressed in this cell line. The cDNA-induced glycine uptake has all the characteristic features of ATB0,+; it is Na+- and Cl−-dependent and is inhibitable by neutral as well as cationic amino acids (results not shown). Thus this experimental system is ideal to examine the interaction of 1-methyltryptophan and other tryptophan derivatives with a cloned ATB0,+. Figure 1(A) describes the effects of tryptophan and its various derivatives on ATB0,+-specific glycine uptake. The uptake was inhibited by L-tryptophan, D-tryptophan, 1-methyl-DL-tryptophan, 4-methyl-DL-tryptophan, 5-methyl-DL-tryptophan, 6-methyl-DL-tryptophan, α-methyl-DL-tryptophan, 5-hydroxy-L-tryptophan and L-tryptophan methyl ester (glycine concentration, 10 μM; inhibitor concentration, 1 mM). In contrast, N-acetyl-L-tryptophan and 5-hydroxytryptamine (serotonin) were not able to inhibit the uptake. ATB0,+ is known to transport many, but not all, of its substrates in L-form and in D-form . We have shown that alanine, serine, methionine, leucine and tryptophan are transported via ATB0,+ in both L- and D-form, while the remaining amino acid substrates are recognized by the transporter only in L-form . The present studies with L-tryptophan and D-tryptophan corroborate these earlier findings. Since 1-methyl-DL-tryptophan was able to inhibit ATB0,+-specific glycine uptake, we compared the inhibitory potencies of the L- and D-isomers of 1-methyltryptophan (results not shown). Even though both racemic forms were able to inhibit the uptake, there was a marked difference in their potencies. 1-Methyl-L-tryptophan was at least 35-fold more potent than 1-methyl-D-tryptophan as an inhibitor of ATB0,+-mediated glycine uptake. The IC50 values for the L- and D-isomers were 23±5 and 780±225 μM respectively.
Interaction of tryptophan and its derivatives with ATB0,+
Interaction of tryptophan derivatives with cloned ATB0,+ expressed heterologously in X. laevis oocytes
The above studies with the mammalian cell expression system have focused on the interaction of 1-methyltryptophan and other tryptophan derivatives with ATB0,+ by examining the effects of these compounds on the transporter-mediated glycine uptake. These studies have shown convincingly that 1-methyltryptophan interacts with ATB0,+, with the L-isomer having much higher affinity for the transporter than the D-isomer. However, these studies have not shown whether or not 1-methyltryptophan is actually a transportable substrate for ATB0,+. It is possible that 1-methyltryptophan inhibits ATB0,+-mediated glycine uptake simply by blocking the interaction of glycine with the substrate-binding site of the transporter, rather than by actually competing with glycine for the transport process. Competition experiments described above do not have the ability to distinguish between transportable substrates and blockers. We wanted to know whether 1-methyltryptophan can actually be transported by ATB0,+. For this, we used the X. laevis oocyte expression system. Since the transport process mediated by ATB0,+ is electrogenic, exposure of ATB0,+-expressing oocytes to transportable substrates will induce an inward current in an Na+- and Cl−-dependent manner. Exposure of the same oocytes to blockers will interfere with currents induced by transportable substrates, but will not induce currents by themselves. This experimental system thus has the ability to differentiate between a transportable substrate and a blocker. Therefore we expressed the cloned mouse ATB0,+ in Xenopus oocytes by injecting the corresponding cRNA, and functional expression of the transporter in the oocytes was confirmed by glycine-induced inward currents. Water-injected oocytes did not show any glycine-inducible currents, indicating that there was no detectable basal electrogenic glycine transport in these oocytes. Once the expression of ATB0,+ was confirmed, we investigated the transport of various tryptophan derivatives via the transporter by monitoring inward currents. Most of the derivatives that inhibited ATB0,+-mediated glycine uptake in mammalian cells showed inward currents in ATB0,+-expressing oocytes (Figure 1B). The magnitude of the currents varied from compound to compound. Importantly, 1-methyl-DL-tryptophan induced marked currents. 5-Hydroxy-L-tryptamine (serotonin) and N-acetyl-L-tryptophan, which did not inhibit ATB0,+-mediated glycine uptake in mammalian cells, did not induce currents in the oocyte expression system. The only notable exception was α-methyl-DL-tryptophan. This derivative was a potent inhibitor of ATB0,+-mediated glycine uptake in mammalian cells, but it failed to induce currents in the oocytes, suggesting that this derivative may be a blocker of ATB0,+. These results showed for the first time that 1-methyl-DL-tryptophan is a transportable substrate for ATB0,+.
Since this experimental approach allows us to monitor directly the actual transport of 1-methyltryptophan via ATB0,+, we performed saturation kinetics for the L- and D-isomers of this derivative and compared the data with those obtained for the L- and D-isomers of tryptophan (Figure 2). The currents induced by all four compounds were saturable in ATB0,+-expressing oocytes. The Michaelis constants for the L- and D-isomers of tryptophan were 26±3 and 288±48 μM respectively. The corresponding values for the L- and D-isomers of 1-methyltryptophan were 93±16 and 462±78 μM respectively. We then analysed the Na+-activation kinetics and Cl−-activation kinetics for 1-methyl-L-tryptophan. This was done by monitoring the inward currents induced by 1 mM 1-methyl-L-tryptophan in the presence of increasing concentrations of Na+ (with Cl− concentration fixed at 100 mM) or Cl− (with Na+ concentration fixed at 100 mM) (Figure 3). The Na+-activation kinetics showed a sigmoidal relationship between Na+ concentration and inward currents. The Hill coefficient (h) was 1.9±0.2, indicating that two Na+ are involved in the activation process. The Michaelis constant (K0.5) for Na+ was 27±2 mM. Similar experiments with Cl− showed a hyperbolic relationship between Cl− concentration and inward currents. The Hill coefficient was 1.2±0.2 and the Michaelis constant was 2.1±0.3 mM. These results show that one Cl− is involved in the activation process. Since 1-methyl-L-tryptophan is zwitterionic under the experimental conditions (pH 7.5), the stoichiometry suggests that the transport process is associated with transfer of one net positive charge into the oocytes per transport cycle. This explains the electrogenic nature of the transport process and the induction of inward currents under voltage-clamp conditions.
Saturation kinetics for ATB0,+-mediated transport of L- and D-isomers of tryptophan and 1-methyltryptophan
Na+- and Cl−-activation kinetics for ATB0,+-mediated transport of 1-methyl-L-tryptophan
α-Methyl-DL-tryptophan as a blocker of ATB0,+
Even though α-methyl-DL-tryptophan was able to inhibit ATB0,+-mediated glycine uptake in HRPE cells (Figure 1A), it failed to induce inward currents in ATB0,+-expressing Xenopus oocytes (Figure 1B). This indicates that this tryptophan derivative may function as a blocker of ATB0,+. We performed additional studies to confirm whether α-methyl-DL-tryptophan is indeed an ATB0,+ blocker. For this, we used the Xenopus oocyte expression system in which the transport function of heterologously expressed human ATB0,+ was monitored by measuring inward currents induced by L-tryptophan under sub-saturating conditions (L-tryptophan concentration, 20 μM). Simultaneous addition of a transportable substrate along with 20 μM L-tryptophan will increase the magnitude of the inward currents, whereas simultaneous addition of a blocker along with 20 μM L-tryptophan will decrease the magnitude of the inward currents. Figure 4(A) describes the results from these experiments. Exposure of ATB0,+-expressing oocytes to 20 μM L-tryptophan induced ∼30 nA inward currents. But when the same oocytes were exposed to L-tryptophan (20 μM) and α-methyl-DL-tryptophan (1 mM) simultaneously, the L-tryptophan-induced currents were blocked completely. This was in contrast with what happened with simultaneous exposure to L-tryptophan and 1-methyl-DL-tryptophan. When 1-methyl-DL-tryptophan was present at 1 mM along with 20 μM L-tryptophan, the magnitude of inward currents was greater than that with 20 μM L-tryptophan alone. This was expected because 1-methyl-DL-tryptophan is a transportable substrate, and when 1 mM of this compound is added to the medium along with L-tryptophan at a sub-saturating concentration (20 μM), the transporter mediates the influx of L-tryptophan as well as 1-methyl-DL-tryptophan, resulting in an increase in the magnitude of inward currents.
Evaluation of α-methyl-DL-tryptophan as a blocker of ATB0,+
These studies with α-methyl-DL-tryptophan as a blocker of ATB0,+ were carried out with L-tryptophan at sub-saturating concentrations. We then wanted to determine the potency of this blocker to interfere with the transporter under conditions that simulate those in vivo, with all naturally occurring amino acids present at normal physiological concentrations. ATB0,+ accepts all proteinogenic amino acids except for glutamate and aspartate. Therefore, using the Xenopus oocyte heterologous expression system, we monitored the transport activity of human ATB0,+ in the presence of an amino acid mixture consisting of 18 different amino acids, each at its respective physiological concentration found in plasma  (Table 1). As expected, inward currents were detected under these conditions in oocytes expressing human ATB0,+ (results not shown). Under similar conditions, the currents induced in water-injected oocytes were less than 5% of the currents observed in ATB0,+-expressing oocytes, demonstrating that the currents observed in ATB0,+-expressing oocytes were almost completely due to the transporter. We then monitored the ability of α-methyl-DL-tryptophan to block the inward currents induced by the same mixture of amino acids in oocytes expressing human ATB0,+ (Figure 4B). There was a dose-dependent increase in the magnitude of inhibition, with maximal inhibition (∼85%) observable at 2.5 mM α-methyl-DL-tryptophan. The concentration of the blocker necessary to elicit 50% of maximal inhibition was 255±24 μM.
|Amino acid||Concentration (μM)|
|Amino acid||Concentration (μM)|
Expression of IDO and ATB0,+ in non-malignant and malignant mammary epithelial cell lines
The studies described so far have shown that 1-methyl-L-tryptophan is a transportable substrate for ATB0,+ and that α-methyl-DL-tryptophan is a blocker of ATB0,+. 1-Methyl-L-tryptophan is an inhibitor of IDO and has been shown to be effective in animal models as a chemotherapeutic agent for the treatment of certain cancers by activation of immune function against tumour cells [24,25]. This tryptophan derivative is currently in clinical trials for evaluation of its efficacy in cancer treatment in humans. It is already known that the ability of 1-methyl-L-tryptophan to inhibit IDO underlies its therapeutic potential as an anticancer agent, but the mechanism of cellular entry of this compound in tumour cells or in tumour-associated immune cells to gain access to its therapeutic target IDO has not been investigated. Since the present studies have shown that ATB0,+ functions as a transporter for 1-methyl-L-tryptophan, we wanted to investigate the relevance of ATB0,+ to the cellular entry of 1-methyl-L-tryptophan and consequent inhibition of intracellular IDO. For this, we needed a cell-culture model system in which IDO and the transporter are co-expressed. We screened nine different mammary epithelial cell lines, three of them being normal non-malignant cell lines (HMEC, HBL100 and MCF10A) and the remaining being malignant cell lines (MCF7, T-47D, ZR-75.1, MDA-MB231, MDA-MB361 and MDA-MB453). Surprisingly, the expression of IDO1 and IDO2 was greater in non-malignant cell lines than in malignant cell lines (Figure 5A). There was little or no expression of these enzymes in malignant cell lines except for the T-47D cell line, which showed appreciable expression of both isoforms of IDO. TDO was expressed at low levels in some cell lines, but there was no malignancy-associated up-regulation of this enzyme. However, ATB0,+ was expressed robustly in three malignant cell lines (MCF7, T-47D and ZR-75.1), while the expression of the transporter was either very low or undetectable in non-malignant cell lines and the remaining three malignant cell lines. This was evident at the level of mRNA expression (Figure 5A) and protein expression (Figure 5B). We confirmed this differential expression of ATB0,+ by monitoring its transport function. The data for glycine uptake in ATB0,+-positive MCF7 cell line and ATB0,+-negative MCF10A cell line are given in Figures 5(C)–5(E). The uptake of glycine was detectable in both cell lines, but only in MCF7 cells the uptake was Na+-dependent, Cl−-dependent and arginine-inhibitable. These are signature characteristics of ATB0,+. Even though glycine uptake in MCF10A was Na+/Cl−-dependent, the inability of arginine to compete with glycine uptake excludes ATB0,+ as the transporter responsible for the uptake. The arginine-sensitive glycine uptake, which is a measure of ATB0,+-specific transport function, is given in Figure 5(E). While the transport function was robust in MCF7 cells, the activity was barely detectable in MCF10A cells, corroborating the data on the differential expression of ATB0,+ in these cells. In subsequent studies, we have shown that GLYT1, which is also an Na+/Cl−-dependent transport system for glycine but is insensitive to arginine inhibition, is responsible for glycine uptake observed in MCF10A cells (results not shown). RT–PCR studies showed that MCF7 cells also express GLYT1 mRNA, but not GLYT2 mRNA (results not shown). However, glycine uptake via GLYT1 should not be inhibitable by arginine. Since most of the glycine uptake in MCF7 cells is arginine-sensitive, we conclude that contribution of GLYT1 to glycine uptake in this cell line is very low.
Expression of IDO1, IDO2, TDO and ATB0,+ in mammary epithelial cell lines
Since glycine serves as a substrate for three different Na+/Cl−-coupled transport systems, namely ATB0,+, GLYT1 and GLYT2, we studied the transport of D-[3H]serine, a known substrate for ATB0,+ with little interaction with the other two transporters , in these cell lines (Figure 6). The uptake of D-serine in MCF7 cells was obligatorily dependent on Na+ and Cl−. The uptake was inhibitable by neutral and cationic amino acids (Figure 7A). Arginine and lysine inhibited the uptake, with IC50 values in the range of 250–500 μM (Figure 7B). These results confirm the expression of ATB0,+ in MCF7 cells. We employed a similar experimental strategy to detect specifically the transport function of ATB0,+ in the remaining seven cell lines. These studies showed that ATB0,+ transport function is detectable only in MCF7, T-47D and ZR-75.1 cells and not in others, corroborating the data on mRNA and protein expression. Since MCF7 cells express ATB0,+ constitutively, we investigated the inhibition of D-serine uptake by 1-methyl-DL-tryptophan, 1-methyl-L-tryptophan and 1-methyl-D-tryptophan to corroborate our earlier findings on the interaction of these compounds with the transporter in heterologous expression systems (results not shown). 1-Methyl-DL-tryptophan was able to inhibit D-serine uptake in a dose-dependent manner in this cell line; the IC50 value for inhibition by 1-methyl-DL-tryptophan was 221±40 μM. When the L- and D-isomers of 1-methyltryptophan were examined individually, it was found that the L-isomer competed with D-serine much more effectively than the D-isomer. The IC50 value for inhibition by the L-isomer was at least 15-fold lower compared with the D-isomer (86±11 μM versus 1580±180 μM). These results show that ATB0,+ interacts with 1-methyltryptophan in a cell line that expresses the transporter constitutively. We also investigated the effect of α-methyl-DL-tryptophan on D-serine uptake in MCF7 cells. The uptake was inhibitable by this blocker of ATB0,+. The inhibition was competitive (Figure 7C). In the absence of the blocker, the values for Kt and Vmax for D-serine uptake were 517±38 μM and 32.4±1.5 nmol/mg of protein per 15 min respectively. The corresponding values were 869±52 μM and 33.3±1.4 nmol/mg of protein per 15 min in the presence of α-methyl-DL-tryptophan (100 μM). Thus the inhibition of D-serine uptake by α-methyl-DL-tryptophan was associated with a decrease in substrate affinity without affecting the maximal velocity.
Features of D-serine uptake in MCF7 cells
Amino acid selectivity and inhibition by α-methyl-DL-tryptophan of D-serine uptake system in MCF7 cells
Evidence for the potential use of α-methyl-DL-tryptophan as a tumour-suppressive agent from its ability to block ATB0,+
The present studies have identified α-methyl-DL-tryptophan as a blocker of ATB0,+ and also have shown that the compound is able to block the transport of amino acids via ATB0,+ under conditions that stimulate those in vivo in terms of amino acid concentrations. We have also shown that the transporter is up-regulated in certain tumour cell lines, but not in all tumour cell lines, and that the expression of the transporter is almost undetectable in non-malignant cell lines. This provides a unique opportunity to examine the potential of α-methyl-DL-tryptophan to deprive ATB0,+-positive tumour cells of essential amino acids and evaluate the consequences. For this, we treated the human mammary epithelial cell lines with 2.5 mM α-methyl-DL-tryptophan in the presence of regular culture medium for 12 days in clonogenic assay and quantified the colony formation for each cell line in the presence and absence of α-methyl-DL-tryptophan (Figure 8). We found no effect of α-methyl-DL-tryptophan on HMEC, HBL100 and MCF10A cells, which are non-malignant and ATB0,+-negative in their ability to form colonies. Similarly, the blocker had little or no effect on colony formation in MDA-MB231, MDA-MB361 and MDA-MB453 cell lines, which are malignant but express no or very low levels of ATB0,+. In contrast, the three cell lines that are malignant and ATB0,+-positive (MCF7, T-47D and ZR-75.1) were affected markedly by α-methyl-DL-tryptophan in the clonogenic assay. At 2.5 mM, the blocker was able to inhibit colony formation by 60–75% in these three cell lines. A dose–response study with MCF7 cells using the same assay conditions gave an IC50 value of 160±15 μM for the inhibition (results not shown).
Influence of α-methyl-DL-tryptophan on the colony-forming ability of mammary epithelial cell lines
Since treatment with α-methyl-DL-tryptophan suppressed colony formation in malignant cell lines in an ATB0,+-dependent manner, we investigated the influence of this treatment on cell cycle to determine which stage of the cycle is affected. We used three different cell lines in this experiment: MCF10A (non-malignant and ATB0,+-negative), MCF7 (malignant and ATB0,+-positive) and MDA-MB453 (malignant and ATB0,+-negative) (Figure 9). Analysis of the progression of cells through cell cycle under control conditions and in the presence of α-methyl-DL-tryptophan showed that the treatment with the blocker had no effect on cell cycle in MCF10A and MDA-MB453 cell lines that do not express ATB0,+. In contrast, the treatment affected the cell cycle progression in the ATB0,+-positive MCF7 cell line. The cells were arrested at G1/G0 stage to a significant extent when treated with α-methyl-DL-tryptophan. This was evident from the relative percentage of cells that were in G1/G0- and S-phases. In the absence of the blocker, the percentage of MCF7 cells in G1/G0- and S-phases was 63±1 and 10±2 respectively. But when the cells were treated with α-methyl-DL-tryptophan for 24 h, the corresponding values were 82±1 and 3±1 respectively. The treatment-induced increase in the percentage of the cells in G1/G0-phase and decrease in the percentage of the cells in S-phase were statistically significant (P<0.001). Similar results were obtained with treatment for 48 and 72 h. These results show that α-methyl-DL-tryptophan prevents MCF7 cells from progressing from G1-phase to S-phase. Since this effect is not seen with MCF10A and MDA-MB453 cells, we conclude that the observed effect in MCF7 cells is due to blockade of ATB0,+ transport function.
Influence of α-methyl-DL-tryptophan on cell-cycle profile of MCF10A, MCF7 and MDA-MB453 cell lines
Interaction of tryptophan derivatives with cloned human LAT1 and expression of the transporter in breast cancer cell lines
LAT1/4F2hc is a heterodimeric amino acid transporter that functions as an obligatory amino acid exchanger [26,27]. LAT1 is the transporter and 4F2hc is necessary for proper targeting of the transporter to the plasma membrane. The activity of LAT1 is not dependent on Na+. Aromatic amino acids such as tryptophan and branched-chain amino acids such as valine are high-affinity substrates for this transporter. The expression of LAT1 is increased in certain tumours, especially in the brain and lung [26,28]. Therefore we investigated the interaction of tryptophan derivatives with cloned human LAT1. For this, we co-expressed human LAT1 cDNA and human 4F2hc cDNA in HRPE cells using the vaccinia virus expression technique and used L-valine as the substrate to monitor the transporter activity. The uptake of valine increased in cDNA-transfected cells by 3-fold compared with vector-transfected cells (Figure 10). In the presence of tryptophan, 1-methyl-L-tryptophan, 1-methyl-DL-tryptophan and α-methyl-DL-tryptophan (1 mM), the cDNA-induced valine uptake was abolished completely, demonstrating that these tryptophan derivatives interact with the transporter.
Inhibition of LAT1 transport function by tryptophan derivatives
Since α-methyl-DL-tryptophan inhibits LAT1 effectively, the question arises as to the role of LAT1 in the ability of this tryptophan derivative to cause growth arrest in certain breast cancer cell lines. Therefore we studied the expression of LAT1 in non-malignant and malignant mammary epithelial cell lines to see if the transporter expression is enhanced in cancer cell lines (Figure 11A). These studies showed that LAT1 is expressed in these cell lines, but there was no difference in the expression levels between non-malignant and malignant cell lines. This was not surprising because enhanced expression of LAT1 in cancer is tissue-specific . Previous studies have shown that LAT1 expression is not altered in breast cancer . Our studies confirm these previous findings. The ability of α-methyl-DL-tryptophan to cause growth arrest is seen only in three malignant mammary epithelial cell lines (MCF7, T-47D and ZR75.1) and not in the non-malignant cell lines (HMEC, HBL100 and MCF10A) and the other three malignant cell lines (MDA-MB231, MDA-MB361 and MDA-MB453). Since the expression of LAT1 is not different among all these nine cell lines, inhibition of LAT1 by α-methyl-DL-tryptophan is not responsible for the observed ability of this tryptophan derivative to block growth arrest in MCF7, T-47D and ZR75.1 cell lines. These findings further support our conclusion that the growth arrest caused by α-methyl-DL-tryptophan in these three cell lines occurs through blockade of ATB0,+ function.
RT–PCR analysis of the expression of LAT1 mRNA in non-malignant and malignant HMEC cell lines (A) and ATB0,+ mRNA in non-malignant and malignant human colon epithelial cell lines (B)
Expression of ATB0,+ in colon cancer cell lines
To determine whether the expression of ATB0,+ is induced in other malignant cell lines, we measured the levels of ATB0,+ mRNA in two non-malignant human colonic epithelial cell lines (NCM460 and CCD841) and nine malignant human colonic epithelial cell lines (SW480, SW620, KM12C, KM12L4, HT29, HCT116, Colo201, Colo205 and LS174T) (Figure 11B). We found robust up-regulation of ATB0,+ expression in five out of nine malignant cell lines. This suggests that the induction of ATB0,+ expression is not unique to certain breast cancer cell lines; the phenomenon is seen also in other malignant cell types.
The present study was started initially to investigate the transport of the IDO inhibitor 1-methyltryptophan via ATB0,+. The results of this investigation have shown that 1-methyltryptophan is indeed a transportable substrate for ATB0,+. However, we could not examine the clinical relevance of these findings with the cell lines employed in the present study, since there was no malignancy-associated up-regulation of IDO in mammary epithelial cell lines. It is possible that IDO is up-regulated mostly in tumour-associated immune cells and stromal cells. Recently, we reported on the expression of ATB0,+ in colon cancer metastasis in the liver . While the expression was robust in metastasized cancer cells, there was a significant up-regulation of the transporter expression in liver cells surrounding the metastasized cancer cells. Based on these findings, we speculated that cancer cells may secrete factors that might act on surrounding non-cancerous cells to induce ATB0,+ expression . Therefore it would be interesting to examine the expression of this transporter in tumour-associated immune cells that exhibit up-regulation of IDO. The present findings that ATB0,+ transports 1-methyl-DL-tryptophan also suggest that the transporter might play a role in the oral bioavailability of this compound. ATB0,+ is expressed in the intestinal tract, especially in the colon . The intestinal/colonic absorption of this drug may be facilitated at least partly by this transporter.
The identification of a tryptophan derivative as a blocker of ATB0,+ was an unexpected and surprising outcome of the present study. α-Methyl-DL-tryptophan blocks the transport function of ATB0,+ without being itself transported. To our knowledge, this is the first compound to be identified as a blocker of this transporter. However, additional studies may be needed to confirm these findings. We have come to the conclusion that α-methyl-DL-tryptophan is not a transportable substrate, solely based on the findings that the compound did not induce detectable inward currents in ATB0,+-expressing oocytes. It is possible that the compound is actually transported via ATB0,+ but the transport process is electroneutral. This possibility is very unlikely, because α-methyl-DL-tryptophan is a zwitterion with a carboxylate group and an amino group, similar to other transportable tryptophan derivatives examined in the study. If this compound is transported via ATB0,+, there is no rational basis to speculate that the transport process may be electrically silent. Comparison of uptake of α-methyl-DL-tryptophan between control oocytes and ATB0,+-expressing oocytes can provide data to demonstrate unequivocally whether or not this tryptophan derivative is a transportable substrate. However, lack of radiolabelled α-methyl-DL-tryptophan from commercial sources makes it difficult to perform such experiments at the present time. Nonetheless, the present studies, although not unequivocal, strongly suggest that α-methyl-DL-tryptophan is most likely a blocker of ATB0,+. However, the potency of this compound to block the transport function of ATB0,+ is weak. With amino acid concentrations simulating in vivo conditions, the compound was able to cause 50% blockage of ATB0,+ transport function only at ∼250 μM.
We were intrigued by the finding that one of the tryptophan derivatives may actually be a blocker of ATB0,+, because specific blockers of this transporter may have potential in cancer chemotherapy. Since tumour cells up-regulate ATB0,+ to meet their increasing demands for amino acids, blockade of the transport function of ATB0,+ may offer an effective means to starve the tumour cells of essential amino acids. ATB0,+-expressing tumour cells may suffer from amino acid deprivation when treated with α-methyl-DL-tryptophan with consequent cell-cycle arrest. The results of the present studies illustrate two important points. First, α-methyl-DL-tryptophan is not toxic to human mammary epithelial cell lines, whether or not the cells are malignant, as long as there is no ATB0,+ contributing to amino acid nutrition. Secondly, α-methyl-DL-tryptophan is able to arrest colony formation in tumour cells in a ATB0,+-specific manner. These results provide the proof-of-concept for the potential of ATB0,+-specific blockers as anticancer agents in tumours that overexpress ATB0,+. Thus the present studies reveal the potential of ATB0,+ as a drug target in cancer treatment. High-affinity blockers of this transporter may have therapeutic use in preventing tumour progression. Since the transporter is expressed at very low levels in most cells under normal conditions and marked up-regulation of the transporter is seen in cancer, blockade of the transport function of ATB0,+ may have a tumour-specific effect, without affecting the surrounding normal cells. The present studies have shown that α-methyl-DL-tryptophan is an inhibitor of the transport function of not only ATB0,+ but also LAT1. Increased expression of LAT1 has been demonstrated in certain cancers but not in all. But there are significant differences between these two transporters which are relevant to their potential as therapeutic targets for cancer treatment. ATB0,+ is highly concentrative with a unique ability to mediate uphill transport of amino acids into cells because of the driving forces involved in the transport process. In contrast, LAT1 is not active. Furthermore, the transport function of LAT1 is bidirectional. It functions as an obligatory amino acid exchanger, a characteristic more suitable for equilibration of amino acids inside the cells in relation to the extracellular milieu than for concentrative influx of amino acids into cells. Cancer cells have a unique need for increased influx of amino acids to support their growth. The functional differences between ATB0,+ and LAT1 highlight the potential of the former as a promising drug target for cancer treatment.