Upregulation of the amino acid transporter ATB0,+ (SLC6A14) in colorectal cancer and metastasis in humans.

ATB(0,+) (SLC6A14) is a Na(+)/Cl(-)-coupled arginine transporter expressed at low levels in normal colon. Arginine is an essential amino acid for tumor cells. Arginine is also the substrate for nitric oxide synthases (NOSs). Since arginine and arginine-derived nitric oxide (NO) play a critical role in cancer, we examined the expression of ATB(0,+) in colorectal cancer. Paired normal and cancer tissues from colectomy specimens of 10 patients with colorectal cancer and from the liver tissue of one patient with hepatic metastasis from a colonic primary were used for the analysis of the levels of ATB(0,+) mRNA, inducible NOS (iNOS) mRNA and the corresponding proteins. Tissues samples from the colon, liver, and lymph nodes of an additional patient with metastatic colon cancer were analyzed for ATB(0,+) protein alone. We also examined the levels of nitrotyrosylated proteins. The ATB(0,+) mRNA increased 22.9+/-3.0-fold in colorectal cancer compared to normal tissue and the increase was evident in each of the 10 cases examined. iNOS mRNA increased 5.2+/-1.1-fold in cancer specimens. The changes in mRNA levels were associated with an increase in ATB(0,+), iNOS, and nitrotyrosylated proteins. The increased expression of ATB(0,+) and iNOS was also demonstrated in liver and lymph node specimens with metastases from colonic primaries. This study strongly suggests that the upregulation of ATB(0,+) may have a pathogenic role in colorectal cancer. Since ATB(0,+) is a versatile transporter not only for arginine but also for several drugs including NOS inhibitors, these findings have significant clinical and therapeutic relevance.

Induction of cystine-glutamate transporter xc- by human immunodeficiency virus type 1 transactivator protein tat in retinal pigment epithelium.

PURPOSE: The transactivator protein Tat encoded by the human immunodeficiency virus-1 (HIV-1) genome reduces glutathione levels in mammalian cells. Because the retina contains large amounts of glutathione, a study was undertaken to determine the influence of Tat on glutathione levels, gamma-glutamyl transpeptidase activity, and the expression and activity of the cystine-glutamate transporter xc- in the human retinal pigment epithelial cell line ARPE-19 and in retina from Tat-transgenic mice. METHODS: The transport function of xc- was measured as glutamate uptake in the absence of Na+. mRNA levels for xCT and 4F2hc, the two subunits of system xc-, were monitored by RT-PCR and Northern blot and protein levels by Western blot. The expression pattern of xCT and 4F2hc in the mouse retina was analyzed by immunofluorescence. RESULTS: Expression of Tat in ARPE-19 cells led to a decrease in glutathione levels and an increase in gamma-glutamyl transpeptidase activity. The transport function of xc- was upregulated, and this increase was accompanied by increases in the levels of mRNAs for xCT and 4F2hc and in corresponding protein levels. The influence of Tat on the expression of xc- was independent of the cellular status of glutathione. Most of these findings were confirmed in the retina of Tat-transgenic mice. CONCLUSIONS: Expression of HIV-1 Tat in the retina decreases glutathione levels and increases gamma-glutamyl transpeptidase activity. Tat also upregulates the expression of system xc-. Glutathione levels may be decreased and the expression of xc- enhanced in the retina of patients with HIV-1 infection, leading to oxidative stress and excitotoxicity.

Up-regulation of the amino acid transporter ATB(0,+) (SLC6A14) in carcinoma of the cervix.

OBJECTIVE: ATB(0,+) is an energy-coupled transporter for arginine and amino acid-based prodrugs. The objective of the study was to examine the expression of this transporter in cervical cancer. METHODS: Specimens of normal ectocervical mucosa and cervical squamous cell carcinoma were used for determination of ATB(0,+) mRNA levels by RT-PCR. A commercial dot blot of paired normal cervix and cervical cancer cDNA was also used to quantify ATB(0,+) mRNA. ATB(0,+) mRNA and protein in tissue sections were analyzed by in situ hybridization and immunohistochemistry/immunofluorescence. RESULTS: ATB(0,+) mRNA increased 5.6-fold (P < 0.0004) in cervical cancer compared to normal cervix. This was associated with a parallel increase in ATB(0,+) protein. CONCLUSIONS: Expression of ATB(0,+) is minimal in normal cervix and is up-regulated in cervical cancer. The up-regulation of this highly concentrative transporter for arginine and prodrugs in cervical cancer has significant clinical and therapeutic relevance.

Transport of amino acid esters and the amino-acid-based prodrug valganciclovir by the amino acid transporter ATB(0,+).

PURPOSE: The purpose of this study was to analyze the transport of amino acid esters and the amino-acid-based prodrug valganciclovir by the Na(+)/Cl(-)-coupled amino acid transporter ATB(0,+). METHODS: The interaction of amino acid esters and valganciclovir with the cloned rat ATB(0,+) was evaluated in a mammalian cell expression system and in the Xenopus oocyte expression system. RESULTS: In mammalian cells, expression of ATB(0,+) induced glycine uptake. This uptake was inhibited by valine and its methyl, butyl, and benzyl esters. The benzyl esters of other neutral amino acids were also effective inhibitors. Valganciclovir, the valyl ester of ganciclovir, was also found to inhibit ATB(0,+)-mediated glycine uptake competitively. Exposure of ATB(0,+)-expressing oocytes to glycine induced inward currents. Exposure to different valyl esters (methyl, butyl, and benzyl), benzyl esters of various neutral amino acids, and valganciclovir also induced inward currents in these oocytes. The current induced by valganciclovir was saturable with a K0.5 value of 3.1+/-0.7 mM and was obligatorily dependent on Na+ and Cl-. The Na+:Cl-:valganciclovir stoichiometry was 2 or 3:1:1. CONCLUSIONS: Amino acid esters and the amino-acid-based prodrug valganciclovir are transported by ATB(0,+). This shows that ATB(0,+) can serve as an effective delivery system for amino acid-based prodrugs.

Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon.

D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine.

Transport of amino acid-based prodrugs by the Na+- and Cl(-) -coupled amino acid transporter ATB0,+ and expression of the transporter in tissues amenable for drug delivery.

We evaluated the potential of the Na(+)- and Cl(-)-coupled amino acid transporter ATB(0,+) as a delivery system for amino acid-based prodrugs. Immunofluorescence analysis indicated that ATB(0,+) is expressed abundantly on the luminal surface of cells lining the lumen of the large intestine and the airways of the lung and in various ocular tissues, including the conjunctival epithelium, the tissues easily amenable for drug delivery. We screened a variety of beta-carboxyl derivatives of aspartate and gamma-carboxyl derivatives of glutamate as potential substrates for this transporter using heterologous expression systems. In mammalian cells expressing the cloned ATB(0,+), several of the aspartate and glutamate derivatives inhibited glycine transport via ATB(0,+). Direct evidence for ATB(0,+)-mediated transport of these derivatives was obtained in Xenopus laevis oocytes using electrophysiological methods. Exposure of oocytes, which express ATB(0,+) heterologously, to aspartate beta-benzyl ester as a model derivative induced inward currents in a Na(+)- and Cl(-)-dependent manner with a Na(+)/Cl(-)/aspartate beta-benzyl ester stoichiometry of 2:1:1. ATB(0,+) transported not only the beta-carboxyl derivatives of aspartate and the gamma-carboxyl derivatives of glutamate but also valacyclovir, which is an alpha-carboxyl ester of acyclovir with valine. The transport of valacyclovir via ATB(0,+) was demonstrable in both heterologous expression systems. This process was dependent on Na(+) and Cl(-). The ability of ATB(0,+) to transport valacyclovir was comparable with that of the peptide transporter PEPT1. These findings suggest that ATB(0,+) has significant potential as a delivery system for amino acid-based drugs and prodrugs.