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Increased L-arginine transport in a nitric oxide-producing metastatic colon cancer cell line
J C Cendan1, W W Souba, E M Copeland
1Department of General Surgery, University of Florida College of Medicine, Gainesville 32610, USA.
Background:
Little is known about amino acid transport in human neoplastic cells. We previously characterized L-arginine transport in the primary human colon cancer cell line, SW480, and found it is principally mediated by the sodium-independent system y+. In this study, we characterized L-arginine transport in the metastatic cell line, SW620, and compared it with that in the primary cell line, SW480.
Methods:
Transport of 3H-L-arginine in cell monolayers was analyzed in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transporter affinity (Km) and maximal transport velocity (Vmax). Transport was further characterized through blockade with known amino acids. In addition, the effect of cell age (i.e., time in culture) on arginine transport was examined at 2 and 9 days after seeding. Cellular proliferation was assessed by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.
Results:
L-Arginine uptake was primarily sodium independent in the SW620 cell line. Kinetic and amino acid-inhibition studies revealed a single high-affinity, sodium-independent L-arginine transporter (Vmax = 1286.3 +/- 158.3 pmol/mg protein/30 s; Km = 46.8 +/- 4.2 microM). Sodium-independent transport was blocked by system y+ substrates L-homoarginine, L-ornithine and L-lysine. Sodium-dependent uptake occurs through a single transporter with system BO,+ characteristics (Km = 16.15 +/- 2.1 microM; Vmax = 329.94 +/- 29.7 pmol/mg protein/30 s). Arginine transport increased with time in culture with day 2 cells transport velocity = 241.7 +/- 33.6 pmol/mg protein/30s, whereas day 9 cells transport velocity = 377 +/- 15.4 pmol/mg protein/30 s (p < 0.01). Cellular-proliferation studies revealed a doubling time of 3.2 days for SW620 and 5.4 days for SW480 (p < 0.05).
Conclusions:
L-Arginine transport in these neoplastic cell lines occurs primarily through sodium-independent, high-affinity system y+. Vmax was increased 180% in the metastatic variant (SW620), suggesting upregulation of the Y+ transporter. The increased Y+ activity may be a mechanism to provide continuous substrate for tumor growth.