Kinetic parameters of 3-[123I]iodo-L-α-methyl tyrosine ([123I]IMT) transport in human GOS3 glioma cells

doi:10.1016/S0969-8051(01)00191-3

Nuclear Medicine and Biology

Volume 28, Issue 3, April 2001, Pages 293-297

https://doi.org/10.1016/S0969-8051(01)00191-3 Get rights and content

Abstract

The radiolabelled amino acid 3-[¹²³I]iodo-L-α-methyl tyrosine ([¹²³I]IMT) is a promising tool for the diagnosis and monitoring of brain tumors using single-photon emission tomography (SPECT). However, little is known about the precise kinetics of [¹²³I]IMT uptake in human glioma cells. The kinetic analysis of [¹²³I]IMT transport in human GOS3 glioma cells yielded a high-affinity apparent Michaelis constant (K_m = 20.1 ± 1.5 μM). The maximum transport velocity (V_max) amounted to 34.8 ± 1.9 nmol/mg protein/10 min. Competitive inhibition experiments revealed that [¹²³I]IMT transport is mediated principally by the sodium-independent system L.

Introduction

Since 1989, 3-[¹²³I]iodo-L-α-methyl tyrosine ([¹²³I]IMT) SPECT has become a successful diagnostic tool in the pre- and postoperative evaluation of patients suffering from brain tumors [1], [14]. It has been shown that [¹²³I]IMT SPECT is valuable in the non-invasive grading of gliomas [12], [34], the delineation of tumor extent [17], [31], [32] and the early detection of tumor recurrence [13]. In-vitro and in-vivo studies have revealed a specific transport of [¹²³I]IMT across the blood-brain barrier [8], [15]. In particular, it has been shown that [¹²³I]IMT uptake in normal brain is saturable, temperature-dependent, ouabain-inhibitable and cross-inhibitable by L-tyrosine [8]. In addition, [¹²³I]IMT is metabolically stable and not incorporated into proteins [14]. Therefore, [¹²³I]IMT uptake is related to amino acid transport, which reflects the metabolic demand of the tissue [14].

Functional studies using in-vitro and in-vivo techniques have led to the identification of a multiplicity of amino acid transport systems [2], [3], [21]. The transport of most neutral amino acids involves several distinct transport systems with overlapping substrate specificities. System L is sodium-independent and serves the uptake of branched-chain and aromatic amino acids [27]. Other transporters require sodium for facilitated amino acid transport, e.g. system A, which prefers less bulky substrates, or system ASC, which can be distinguished from system A by its lack of reactivity with N-methyl substrates [2].

Competition studies in patients have suggested a crucial role of the sodium-independent system L for [¹²³I]IMT uptake into human gliomas [15], [16]. In-vitro studies have shown that system L is the main contributor to the uptake of [¹²³I]IMT in human 86HG-39 glioma cells [18]. In addition, [¹²³I]IMT transport in rat C6 glioma cells is mediated largely by the system L and, to a minor extent, by the sodium-dependent system B^0,+ for neutral and cationic amino acids [23]. Despite the iodine label, the apparent Michaelis constant (K_m) and the maximum transport rate (V_max) of [¹²³I]IMT uptake in rat C6 glioma cells were in the same range as those of naturally occurring amino acids [25], [28]. To compare the results obtained in rats with those in human glioma cells, the transport kinetics of [¹²³I]IMT were studied in the recently established human GOS3 glioma cell line [4].

Section snippets

Chemicals and reagents

[¹²³I]IMT was synthesized according to the method of Krummeich et al. [11]. The amino acids L-leucine, L-isoleucine, L-phenylalanine, L-arginine, L-lysine, and L-alanine, α-(methylamino)-isobutyric acid (MeAIB), 2-aminobicyclo[2.2.1]-heptane-2-carboxylic acid (BCH) and Krebs-Ringer bicarbonate buffered medium were purchased from Sigma-Aldrich (Germany). In sodium-free buffer, equimolar amounts of choline chloride and choline bicarbonate replaced NaCl and NaHCO₃, respectively. Dulbecco’s

Time course of [¹²³I]IMT uptake

Fig. 1 represents [¹²³I]IMT uptake in human GOS3 glioma cells as a function of time. [¹²³I]IMT uptake increased rapidly over the initial 10 minutes and approached a transport plateau after 15 minutes. An incubation period of 10 minutes was subsequently chosen for kinetic studies [29].

Transport kinetics of [¹²³I]IMT uptake

[¹²³I]IMT uptake in human GOS3 glioma cells was measured over a range of [¹²³I]IMT concentrations (2.5–50 μM). Non-linear regression analysis of the saturation curve revealed the preponderance of a single

Discussion

[¹²³I]IMT SPECT is a promising diagnostic tool in patients with brain tumors. It is valuable for the non-invasive grading of gliomas [12], [34], the delineation of tumor extent [17], [31], [32] and the early detection of tumor recurrence [13]. To date, [¹²³I]IMT is the only amino acid which has been successfully applied in clinical SPECT studies. In addition, [¹²³I]IMT SPECT is less expensive and more widely available than PET using [¹¹C-methyl]-L-methionine or O-(2-[¹⁸F]fluoroethyl)-L-tyrosine

Conclusion

In accordance with [¹²³I]IMT transport in rat C6 glioma cells, [¹²³I]IMT uptake in human GOS3 glioma cells is handled principally via the sodium-independent system L. In addition, the kinetic parameters of [¹²³I]IMT uptake in both cell lines are in the range of those of naturally occurring amino acids.

Acknowledgements

The authors gratefully acknowledge the technical assistance of Mrs. Mara Wachsmuth and Mrs. Ruth Laumann (Department of Neurology); and the continuous and ongoing support of Prof. T. Kuwert (Department of Nuclear Medicine, Friedrich-Alexander-Universität, Erlangen, Germany) and Prof. K.-J. Langen (Institute of Medicine, Research Center of Jülich, Germany). The study has been presented as a poster at the SNM-meeting in St. Louis and has been assigned the Berson-Yalow-Award.

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The l-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and l-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.
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We examined 3-[¹²³I]iodo-α-methyl-l-tyrosine ([¹²³I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure.
Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [¹²⁵I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [¹²⁵I]IMT uptake were examined.
Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B⁰AT was not detected. [¹²⁵I]IMT uptake in DLD-1 cells involved Na⁺-independent system L primarily and Na⁺-dependent system(s). Uptake of [¹²⁵I]IMT in Na⁺-free buffer followed Michaelis–Menten kinetics, with a K_m of 78 μM and V_max of 333 pmol/10⁶ cells per minute. Neutral d- and l-amino acids with branched or aromatic large side chains inhibited [¹²⁵I]IMT uptake. Tyrosine analogues, tryptophan analogues, l-phenylalanine and p-halogeno-l-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-l-phenylalanine (l-DOPA), dl-threo-β-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-l-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [¹²⁵I]IMT uptake, but l-tyrosine methyl ester and R(+)/S(−)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [¹²⁵I]IMT uptake except l-serine and d/l-cysteine.
[¹²⁵I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.
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¹²³I-Iodo-α-methyltyrosine (IMT) transport into lymphomas has not been fully characterized. In rat Nb2-11C and Nb2-Sp lymphoma cell lines, linear uptake of ¹²³I-IMT occurred rapidly within 5–10 min. Eadie–Hoftee plots of ¹²³I-IMT uptake gave apparent K_m's of 8.34±1.17 and 9.64±1.05 μM for Nb2-11C and Nb2-Sp cells, respectively, and involved the L and B^0,+ systems. In lymphoma-bearing rats, injected ¹²³I-IMT accumulated rapidly in the primary tumors but gave a low tumor-to-background ratio of 2:1. ¹²³I-IMT was transported rapidly into lymphoma cells both in vitro and in vivo, but low target-to-nontarget ratio may not make ¹²³I-IMT practical for scanning in vivo.
Efficient and cost-effective experimental determination of kinetic constants and data: The success of a Bayesian systematic approach to drug transport, receptor binding, continuous culture and cell transport kinetics
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Details about the parameters of kinetic systems are crucial for progress in both medical and industrial research, including drug development, clinical diagnosis and biotechnology applications. Such details must be collected by a series of kinetic experiments and investigations. The correct design of the experiment is essential to collecting data suitable for analysis, modelling and deriving the correct information. We have developed a systematic and iterative Bayesian method and sets of rules for the design of enzyme kinetic experiments. Our method selects the optimum design to collect data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. The rules select features of the design such as the substrate range and the number of measurements. We show here that this method can be directly applied to the study of other important kinetic systems, including drug transport, receptor binding, microbial culture and cell transport kinetics. It is possible to reduce the errors in the estimated parameters and, most importantly, increase the efficiency and cost-effectiveness by reducing the necessary amount of experiments and data points measured.
Characterization of 3-[125I]iodo-α-methyl-L-tyrosine transport via human L-type amino acid transporter 1
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We examined transport of 3-[¹²⁵I]iodo-α-methyl-L-tyrosine ([¹²⁵I]IMT) in Xenopus laevis oocytes co-expressing human L-type amino acid transporter 1 (a component of system L) and human 4F2hc. Human LAT1 mediated transport of [¹²⁵I]IMT. [¹²⁵I]IMT uptake was decreased by the presence of L-isomers of Cys, Leu, Ileu, Phe, Met, Tyr, His, Trp and Val and D-isomers of Leu, Phe and Met. Human LAT1-mediated [¹²⁵I]IMT uptake was highly stereoselective for the L-isomers of Tyr, His, Trp, Val and Ileu. To examine the effects of 3-iodination and α-methylation on IMT transport, kinetic parameters of IMT were compared with those of mother Tyr and 3-[¹²⁵I]iodo-L-tyrosine (3-I-Tyr). Uptake of Tyr, 3-I-Tyr and [¹²⁵I]IMT followed Michaelis-Menten kinetics, with K_m values of 29.0 ± 5.1, 12.6 ± 6.1 and 22.6 ± 4.1 μM, respectively. Neither the α-methyl group nor the size of the 3-iodinated Tyr residue was an obstacle to transport via hLAT1. Furthermore, affinity of IMT for hLAT1 is higher than that of the natural parent tyrosine. The level of efflux mediated by hLAT1 was highly stimulated by extracellularly applied L-Leu, suggesting exchange of [¹²⁵I]IMT and L-Leu via hLAT1.
3-[123I]Iodo-α-methyl-L-tyrosine: Uptake mechanisms and clinical applications
2002, Nuclear Medicine and Biology
3-[¹²³I]Iodo-α-methyl-L-tyrosine (IMT) is an artificial amino acid which has gained considerable interest in Nuclear Medicine in the last two decades. Although the tracer is not incorporated into proteins it exhibits high uptake in brain tumors and appears to be a valuable tool especially for the diagnostic evaluation and therapy planning of patients with cerebral gliomas. In this paper the present knowledge of the uptake mechanisms and the clinical applications of IMT are reviewed and the clinical perspectives discussed.

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Nuclear Medicine and Biology

Abstract

Introduction

Section snippets

Chemicals and reagents

Time course of [¹²³I]IMT uptake

Transport kinetics of [¹²³I]IMT uptake

Discussion

Conclusion

Acknowledgements

References (34)

Role of oxoproline in the regulation of neutral amino acid transport across the blood-brain barrier, J Biol Chem

Protein measurement with the folin phenol reagent, J Biol Chem

Identification and functional characterization of a Na⁺-independent neutral amino acid transporter with broad substrate selectivity, J Biol Chem

Neutral amino acid transport systems in chinese hamster ovary cells, J Biol Chem

Evidence of two Na⁺-independent neutral amino acid transport systems in primary cultures of rat hepatocytes, J Biol Chem

Role of amino acid transport and countertransport in nutrition and metabolism, Phys Rev

Transporters for cationic amino acids in animal cells: discovery, structure and function, Phys Rev

Growth inhibition of newly established glioma cell lines by leukemia inhibitory factor, J Neurooncol

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Stimulation of <sup>125</sup>I-3-iodo-α-methyl-l-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters

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Characterization of <sup>123</sup>I-iodo-α-methyltyrosine transport in rat lymphoma cells

Efficient and cost-effective experimental determination of kinetic constants and data: The success of a Bayesian systematic approach to drug transport, receptor binding, continuous culture and cell transport kinetics

Characterization of 3-[<sup>125</sup>I]iodo-α-methyl-L-tyrosine transport via human L-type amino acid transporter 1

3-[<sup>123</sup>I]Iodo-α-methyl-L-tyrosine: Uptake mechanisms and clinical applications

Kinetic parameters of 3-[123I]iodo-L-α-methyl tyrosine ([123I]IMT) transport in human GOS3 glioma cells

Abstract

Introduction

Section snippets

Chemicals and reagents

Time course of [123I]IMT uptake

Transport kinetics of [123I]IMT uptake

Discussion

Conclusion

Acknowledgements

Role of oxoproline in the regulation of neutral amino acid transport across the blood-brain barrier, J Biol Chem

Protein measurement with the folin phenol reagent, J Biol Chem

Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity, J Biol Chem

Neutral amino acid transport systems in chinese hamster ovary cells, J Biol Chem

Evidence of two Na+-independent neutral amino acid transport systems in primary cultures of rat hepatocytes, J Biol Chem

Role of amino acid transport and countertransport in nutrition and metabolism, Phys Rev

Transporters for cationic amino acids in animal cells: discovery, structure and function, Phys Rev

Growth inhibition of newly established glioma cell lines by leukemia inhibitory factor, J Neurooncol

Kinetic parameters of 3-[¹²³I]iodo-L-α-methyl tyrosine ([¹²³I]IMT) transport in human GOS3 glioma cells

Time course of [¹²³I]IMT uptake

Transport kinetics of [¹²³I]IMT uptake

Identification and functional characterization of a Na⁺-independent neutral amino acid transporter with broad substrate selectivity, J Biol Chem

Evidence of two Na⁺-independent neutral amino acid transport systems in primary cultures of rat hepatocytes, J Biol Chem