Neurochemistry International 43 (2003) 501–507
Glutamine transport in C6 glioma cells shows ASCT2 system characteristics Monika Doli´nska a , Anna Dybel a , Barbara Zabłocka b , Jan Albrecht a,∗ a b
Department of Neurotoxicology, Medical Research Centre, Polish Academy of Sciences, Pawi´nskiego St. 5, 02-106 Warsaw, Poland Laboratory of Molecular Biology, Medical Research Centre, Polish Academy of Sciences, Pawi´nskiego St. 5, 02-106 Warsaw, Poland Received 14 October 2002; accepted 8 November 2002
Abstract Previous studies from this laboratory have shown that glutamine (Gln) uptake in a rat astrocytoma-derived C6 cell line shows characteristics similar with the uptake of a model ASC system substrate, threonine, whose pH-dependence and partial tolerance of Li+ substitution for Na+ resemble the ASCT2 variant of the system. In support of the previous findings, RT-PCR analysis revealed that C6 cells strongly express ASCT2 mRNA, but not at all GlnT mRNA or NAT2 mRNA, the A and N system variants specifically engaged in Gln transport in normal CNS. Other features of Gln transport in C6 cells indicating the involvement of ASCT2 system included its resistance to ouabain and stimulation of Gln efflux from the cells in the presence of excess Gln or cysteine (Cys), demonstrating that the system operates in the exchange mode. Replacement of NaCl in the incubation medium with isoosmotic sucrose did neither significantly affect the kinetics, nor any other major characteristics of Gln or Thr transport, including its pH-dependence, inhibition by ASCT system substrates or resistance to the model system A substrate—N-methylamino-isobutyric acid (MeAiB). © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Glutamine uptake; Threonine uptake; Glutamate uptake; C6 cells; System ASCT2; Sodium-dependence; Chloride-dependence
1. Introduction Glutamine (Gln) is a key factor in the growth and metabolism of neoplastic tissues. Experimental studies with tumor cell lines derived from non-CNS and CNS tissues have demonstrated that Gln is a key determinant of tumor growth rate: the cells adapt very slowly, if at all, to Gln-deplete media (Collins et al., 1998; Albrecht et al., 2001; Pawlik et al., 2000; Wasa et al., 1996, 2002). In CNS-derived tumors and tumor cell lines, Gln is predominantly metabolized to glutathione (Portais et al., 1993; Schafer et al., 2001) and glutamate (Portais et al., 1996). Glutathione is believed to account for tumor chemoresistance and radioresistance (Miura and Sasaki, 1991; Bates et al., 1994; Grant and Ironside, 1995; Nagane et al., 1995). Glu, which accumulates in the peritumoral space due both to excessive release (Ye and Sontheimer, 1999) and decreased reuptake (Ye et al., 1999), contributes to necrosis of the neighboring CNS tissues and seizures, both effects being associated with excessive activation of NMDA receptors (Rzeski et al., 2001; Rothstein and Brem, 2001). ∗
Corresponding author. Tel.: +48-22-668-5323; fax: +48-22-668-5532. E-mail address:
[email protected] (J. Albrecht).
Because Gln synthesis in most neoplastic tissues is too low to meet the demand for this amino acid, Gln uptake is widely recognized as rate limiting for Gln utilization (Wasa et al., 1996; Martin et al., 1998; Collins et al., 1998). Gln uptake to tumor cells of different tissue origin is predominantly mediated by system ASC or its human variant, ATB0 (Wasa et al., 1996; Bode et al., 1998). With regard to CNS tissue-derived tumors, the involvement of system ASC has been confirmed by our group for the rat astrocytoma-derived C6 cell line (Doli´nska et al., 2000; Albrecht et al., 2001). The characteristics of Gln uptake, and to a considerable degree the uptake of a model ASC system substrate, threonine (Thr) were compatible with the ASCT2 variant of the system (Table 1), and ASCT2 mRNA was found to be expressed in these cells (Albrecht et al., 2001). Recently, expression of ASCT2 mRNA and system ASC-compatible Gln uptake characteristics were described in a neuroblastoma cell line (Wasa et al., 2002). While system ASCT2 is variably expressed in the brain in site (Utsunomiya-Tate et al., 1996; Broër et al., 1999), it abounds in cultured astrocytes, where it has been characterized in much detail (Nagaraja and Brookes, 1996; Broër et al., 1999, 2000). In this study, we extended the characterization of Gln transport in C6 cells with respect to
0197-0186/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0197-0186(03)00040-8
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Table 1 Comparative characteristics of Gln and Thr uptake in C6 glioma cells Gln uptakea,b Sodium replacement by choline pH-dependence Competitors Sodium replacement by lithium System compatibility a b
Thr uptakeb
No Moderate Variable Thr, Gln, Ser, Cys Partial No ASCT2 ASCT2 (?)
Doli´nska et al. (2000). Albrecht et al. (2001).
the relative role of system ASCT2 and its similarities with the system described in cultured astrocytes. We compared the relative expression of ASCT2 mRNA and of mRNA’a of the A and N system transporters shown to specifically mediate Gln transport in normal CNS. Next we analyzed in detail the dependence of the uptake on different replacements of Na+ or Cl− ions in the medium. We also assessed the ability of the system to operate in the exchange mode. The uptake of Gln was compared with the uptake of Thr.
2. Experimental procedures 2.1. Culturing of C6 cells Rat astrocytoma C6 cells (ATCC, passages 70–80) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% (v/v) fetal calf serum (Gibco) and supplemeted with penicillin (50 U/ml) and streptomycin (50 U/ml) at 37 ◦ C in a humidified atmosphere of 90% air and 10% CO2 , exactly as earlier described (Doli´nska et al., 2000; Albrecht et al., 2001). Cultivation was in 24-well plates for the uptake and efflux studies, and in 35 mm dishes for RNA isolation. 2.2. Amino acid uptake The uptake of radiolabeled Gln, Thr and glutamate (Glu) to C6 cells was measured following a 2 min incubation of the radiolabeled amino acids with cultures grown in 24-well plates exactly as described previously (Doli´nska et al., 2000; Albrecht et al., 2001). Standard incubation media (SIM) contained 150 mM NaCl, 3 mM KCl, 2 mM CaCl2 , 0.8 mM MgCl2 , 5 mM glucose, 10 mM HEPES, pH 7.4 and 0.25 Ci of either l-[G-3 H]Gln, l-[3 H]Thr or l-[G-3 H]Glu (Amersham, UK), supplemented with unlabeled Gln, Thr and Glu, respectively. In NaCl-free media (SIM without NaCl), NaCl was replaced by 250 mM sucrose, and in the legends to figures the media are referred to as “sucrose”. For competition experiments, the concentrations of unlabeled Gln or Thr were 0.078 mM and competing amino acids were added at 64-fold excess, together with radiolabeled amino acids. The concentrations of unlabeled Gln
or Thr used for kinetic analysis of the uptake were as described in the figures. The concentration of Glu in the incubation mixtures was 0.2 mM. The reaction was stopped with 2 ml of ice-cold choline chloride-containing SIM and the cells were lysed by incubation in 1N NaOH at 37 ◦ C for 30 min. 2.3. Amino acid release Cells were preincubated with 0.3 Ci of l-[G-3 H]Gln in SIM for 10 min at 37 ◦ C. After preincubation the medium was removed, the plates were washed four times with 0.5 ml SIM without addition of amino acids and the washes were discarded. The media were then replaced with either SIM or sucrose (SIM-NaCl) media containing no amino acid (“control”), or 5 mM Cys or Gln, respectively. The incubations were carried out each for 10 min, with five replacements of the medium at 2 min intervals. The eluates were pooled for radioactivity measurements. 2.4. RT-PCR analysis Total RNA was isolated from C6 cells or rat cerebral cortex using TRIzol reagent (Life Technologies) and total RNA (5 g) was reverse transcribed using the Superscript II and oligo (dT)12–18 primers (Gibco/BRL). Each cDNA sample (2 l) was then amplified by PCR using the following primers for rat amino acid transporters: ASCT2, 5 -gccagtccacggccaagatc-3 and 5 -gcctggtcgtgttcgctata-3 (Broër et al., 1999); GlnT, 5 -cgcccagtccacggccaagatc-3 and 5 -cagtgaatagagatgcccgag-3 (Varoqui et al., 2000); NAT2, 5 -ttcagcctggtacgtcgatg-3 and 5 -tgacatactttggtgtgcacg-3 (Gu et al., 2001); SAT2, 5 -cccattgtcactgctgagaaa-3 and 5 -tccctgatagtggggacaaa-3 (Yao et al., 2000). The expression was quantitatively related to that of a gene coding for a constitutive protein, GAPDH, for which the primers used were: 5 -tgaaggtcggagtcaacggatttgg-3 and 5 -catgtaggcatgaggtccaccac-3 (Schreiber and Durre, 1999). Following 30 cycles of amplification (1 min at 94 ◦ C, 1 min at 55 ◦ C, and 1 min at 72 ◦ C using the MJ Research thermal cycler PTC-100) the PCR products were resolved on 1.2% agarose gel in an ethidium bromide-containing buffer, with 687 bp ASCT2, 151 bp GlnT, 241 bp NAT2 and 679 bp SAT2 run as standards. The bands were recorded using the Nucleovision system (Nucleotech) and densitometric analysis was carried out using the GelExpert 4.0 program. For sequence analysis, the bands were extracted from agarose using DNA clean-up kit (Akor Laboratories). The analysis was performed for us in the Laboratory of DNA Sequencing, Institute of Biochemistry and Biophysics, Polish Academy Sciences, Warsaw. 2.5. Protein assay Protein content was determined in the NaOH lysates. Protein in the cells used for uptake experiments was
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assayed directly in the plates by the method of Bradford (1976).
Table 2 Relative expression of mRNA’s coding for system ASC, A and N amino acid transporters
2.6. Statistical analysis Statistical significance of the differences between the results was computed using one way ANOVA test, either from Sigma Plot 5.0 or GraphPad Prism 3.02.
503
C6 standard culture Rat cerebral cortex
ASCT2
GlnT/SAT1
NAT2
SAT2
0.45 ± 0.06
nd
nd
0.32 ± 0.05
0.06 ± 0.01
0.77 ± 0.03
0.66 ± 0.05
0.19 ± 0.02
Results are expressed as fraction of GAPDH mRNA and are mean ± S.D. for four to six experiments; nd: not detected.
3. Results Results of the analysis of relative expression of mRNA’s coding for the different Gln-transporting proteins in C6 cells and in rat cerebral cortex are shown in Table 2. ASCT2 mRNA abounds in C6 cells, and is relatively weakly expressed in rat cerebral cortex. By contrast, the RNAs of the A system Gln transporting protein, GlnT/SAT1 (Varoqui et al., 2000; Armano et al., 2002) and of the Gln-specific system N transporter NAT2 (Gu et al., 2001) which, as expected, are strongly expressed in the rat cerebral cortex, were not detected in C6 cells. SAT2 mRNA, which codes for an ubiquitous A system showing no preference for Gln (Armano et al., 2002) was expressed both in C6 cells and rat cerebral cortex. Effects of different modifications of composition of the medium on Gln and Thr uptake are illustrated in Fig. 1. In accord with the previously reported data (Doli´nska et al., 2000; Albrecht et al., 2001), both Gln and Thr uptake were inhibited by substitution of Na+ with Li+ or choline, whereby Gln uptake activity was relatively less sensitive to replacement by Li+ than Thr uptake. Gln uptake, but not Thr up-
take, was sensitive to Na+ substitution by Tris. Gln uptake, but not Thr uptake was ∼20% inhibited by substitution of Cl− with gluconate. Neither Gln nor Thr uptake was affected by increasing K+ , absence of Ca2+ from the medium or addition of ouabain. Of note, neither Gln nor Thr uptake was significantly inhibited by replacement of NaCl with sucrose. Kinetic analysis revealed that the sucrose replacement decreased by only 25% the Vmax of Gln uptake (Fig. 2; data in the legend), and increased by ∼40% the Vmax of Thr uptake (Fig. 3; data in the legend). Irrespective of whether conducted in a NaCl-containing (“NaCl”) or a NaCl-free, sucrose-containing medium (“sucrose”), both Gln (Fig. 4) and Thr (Fig. 5) uptake was inhibited by system ASC substrates but not by a model system A substrate, MeAiB. The ASC system-compatible Gln and Thr uptake was approximately twice as high at pH 8.0 than at pH 6.0, irrespective of whether tested in the “NaCl” or “sucrose” medium (Fig. 6). The activity of the MeAiB + Thr-resistant uptake of Gln, and the MeAiB + Gln-resistant uptake of Thr, both ascribable to system N, was not more than 10–15% of the system ASC-mediated uptake activity, and showed neither
Fig. 1. Effects of changes in the incubation media on l-[3 H]glutamine and l-[3 H]threonine uptake to C6 cells. Results are mean ± S.D. for three to nine experiments. (∗) P < 0.05 vs. NaCl.
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Fig. 2. Michaelis–Menten plot of the kinetics of l-[3 H]glutamine uptake to C6 glioma cells in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with sucrose (“sucrose”). Results are mean ± S.D. for three to nine experiments. The kinetic parameters for the uptake calculated from the plots are: “NaCl”—Vmax = 14.7 ± 0.6 nmol/(min mg), Km = 0.47 ± 0.07 mM; “sucrose”—Vmax = 10.9 ± 0.6 nmol/(min mg), Km = 0.36 ± 0.07 mM.
pH-dependence nor any change with the replacement of NaCl with sucrose (Fig. 6). Incubation of C6 cells with an excess of Gln or Cys in either the NaCl or sucrose medium stimulated in the same degree the release of newly loaded radiolabeled Gln from the cells (Fig. 7). In contrast to Gln or Thr uptake, the uptake of glutamate (Glu) in C6 cells was depressed in an equal degree by substitution of NaCl with sucrose or choline chloride, and was moderately reduced upon replacement of NaCl with KCl, removal of calcium ions from the media, and addition of a glutamate uptake inhibitor, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (Fig. 8).
Fig. 3. Michaelis–Menten plot of the kinetics of l-[3 H]threonine uptake to C6 glioma cells in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with sucrose (“sucrose”). Results are mean ± S.D. for three to nine experiments. The kinetic parameters for the uptake calculated from the plots are: “NaCl”—Vmax = 11.2 ± 0.6 nmol/(min mg), Km = 0.29 ± 0.07 mM; “sucrose”—Vmax = 15.6 ± 1.1 nmol/(min mg), Km = 0.77 ± 0.09 mM.
Fig. 4. Effects of system ASC substrates and a model system A substrate (MeAiB) on l-[3 H]glutamine uptake to C6 glioma cells in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with sucrose (“sucrose”). Results are mean ± S.D. for five to nine experiments.
Fig. 5. Effects of system ASC substrates and a model system A substrate (MeAiB) on l-[3 H]glutamine uptake to C6 glioma cells in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with sucrose (“sucrose”). Results are mean ± S.D. for five to nine experiments.
4. Discussion The present study confirmed and further supported the earlier evidence (Doli´nska et al., 2000; Albrecht et al., 2001) that Gln transport in C6 glioma cells is predominantly mediated by system ASCT2. ASCT2 mRNA was abundantly expressed in these cells, whereas Gln T mRNA and NAT2 mRNA, coding for the proteins carrying out the A and N system-mediated Gln transport in the CNS, respectively (Varoqui et al., 2000; Gu et al., 2001), were not detected. This expression pattern was thus opposite to that found in the rat cerebral cortex, where the low ASCT2 mRNA levels detected were in accord with previous reports (Utsunomiya-Tate et al., 1996; Broër et al., 1999). Accordingly, a model system A substrate, MeAiB did not inhibit Gln uptake in C6 cells, and the fraction of uptake resistant to inhibition by ASC system substrates and possibly mediated by one of the system N variants (see Doli´nska et al., 2000 for a discussion), did not exceed 15% of the total uptake. Except for moderate lithium tolerance and slight inhibition by replacement of chloride ions with gluconate, the characteristics of Gln uptake matched those of the uptake of a model ASCT substrate, Thr. Insensitivity of Gln and
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Fig. 6. Effect of pH on l-[3 H]glutamine and l-[3 H]threonine uptake to C6 cells measured in the presence of 64-fold excess of MeAiB (“ASC”), and of MeAiB + Thr (Gln uptake) or MeAiB + Gln (Thr uptake) (“N”), in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with sucrose (“sucrose”). Results are mean ± S.D. for four to eight experiments.
Fig. 7. Release of l-[3 H]glutamine from C6 cells superfused with a standard medium (“control”), or with a medium containing 5 mM glutamine (Gln) or cysteine (Cys), in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with sucrose (“sucrose”). Results are mean ± S.D. for four to eight experiments. (∗) P < 0.05 vs. control.
Fig. 8. Effects of changes in the incubation media on l-[3 H]glutamate uptake to C6 cells. Results are mean ± S.D. for three to nine experiments. Metabolic inhibitors were added at the following concentrations: niflumic acid (NiF)—200 M; l-trans-2,4-pyrrolidine dicarboxylate (PDC)—100 M. (∗) P < 0.05 vs. NaCl.
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Thr uptake to the Na/K ATPase inhibitor, ouabain, indicated that the uptake is not electrogenic, further excluding system A-mediated transport (McGivan and Pastor-Anglada, 1994; Sugawara et al., 2000). Gln release from the cells was stimulated by Gln and an ASC substrate, Cys, an observation consistent with the propensity of system ASCT2 to operate in the exchange mode (Broër et al., 1999, 2000). The most striking observation of the present study was that the major characteristics of the ASCT2-system mediated Gln or Thr uptake: its kinetics, pH-dependence and competition by system ASC substrates, but also homo- and heteroexchange of Gln, were not significantly affected by isoosmotic replacement of sodium chloride in the incubation medium with sucrose. To see whether the tolerance of a sodium chloride-free medium is a specific feature of Gln and Thr uptake, we looked at the characteristics of Glu uptake, a process well characterized in C6 cells. The 50% inhibition of Glu uptake upon the replacement of sodium chloride by choline chloride and sucrose alike (Fig. 8), is consistent with the magnitude of the sodium-independent component of Glu uptake reported in C6 cells by Cho and Bannai (1990), and in other glioma cell lines by Ye et al. (1999). In accordance with the relatively active sodium-independent Glu uptake in C6 cells, PDC, a strong inhibitor of sodium-dependent uptake in C6 cells (Dowd et al., 1996), only moderately inhibited the total Glu uptake. A partial inhibition by the chloride channel blocker, niflumic acid (NIF) is likewise a typical feature of sodium-independent Glu uptake in cultured CNS cells (reviewed by Anderson and Swanson, 2000), and so is its partial inhibition by K+ ions and in Ca2+ -free media (Faivre-Bauman et al., 1974; Nicklas and Browning, 1983). Hence, the C6 cell line used in the present study behaved in a typical way with regard to Glu transport. Non-depressed Gln or Thr uptake in a sucrose medium deplete of sodium and chloride is difficult to reconcile with the inhibition of the uptake in media in which sodium chloride was replaced by choline or lithium chloride and in the case of Gln uptake, also by Tris. Since the apparent Km value for Na+ of ASCT2 is very low (∼5 mM; Utsunomiya-Tate et al., 1996; Broër et al., 2000), we speculated that this may have been due to the fact that the ion strength of the sucrose-containing medium was not high enough to release Na+ from its binding site, rendering it apparently Na+ -independent. Consistent with this interpretation, oocytes expressing the ASCT2 carrier took up Gln with 50% efficiency even in a medium deplete of NaCl, following extensive washing with sucrose (Broër, personal communication). Recently, inhibition by sodium replacement with choline, but stimulation in media in which sodium chloride was changed to isoosmotic sucrose, has been found to characterize the uptake of polyamines to astrocytes (Dot et al., 2000) and a glioma cell line (Molderings et al., 2001). The interpretation offered in the latter cases was that the respective cations acted as inhibitors of the uptake. In normal rat astrocytes in situ, Gln transport is mediated by a system N protein, SN1, whose activity and the direc-
tion of transport (inward or outward) is regulated by pH and sodium gradients (Chaudhry et al., 1999; Broër et al., 2002). This ion-coupled Gln transport is one of the means by which glutamine availability is controlled to meet the varying metabolic demands in the different regions of the CNS (Boulland et al., 2002). Amino acid competition characteristics of Gln uptake have suggested that in neoplastic CNS and non-CNS tissues including solid tumors, Gln transport is ubiquitously mediated by system ASC (see Section 1 for references). If the transport in glial tumors in situ involves the ASCT2 transporter, is sodium-and chloride independent and relatively weakly pH dependent as it is in the C6 cell line, this could result in significant disturbances in the various aspects of glutamine metabolism in the tumor-affected regions of the brain.
Acknowledgements We thank Stefan Broër for valuable suggestions, and Małgorzata Bogaci´nska, Inez Fr˛es´ko and Mirosława Poławska for technical assistance. The study was supported by SCSR grant no. 6P05A00321.
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