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Increasedl-arginine transport in a nitric oxide-producing metastatic colon cancer cell line

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Abstract

Background: Little is known about amino acid transport in human neoplastic cells. We previously characterizedl-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 characterizedl-arginine transport in the metastatic cell line, SW620, and compared it with that in the primary cell line, SW480.

Methods: Transport of3H-l-arginine in cell monolayers was analyzed in the presence and absence of sodium. Kinetic studies were performed over a range ofl-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 asssessed 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-independentl-arginine transporter (Vmax=1286.3 ± 158.3 pmol/mg protein/30 s; Km=46.8 ± 4.2 µM). Sodium-independent transport was blocked by system y+ substratesl-homoarginine,l-ornithine andl-lysine. Sodium-dependent uptake occurs through a single transporter with system BO,+ characteristics (Km=16.15 ± 2.1 µM; 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.

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Authors and Affiliations

  1. Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Wiley W. Souba MD, ScD

  2. Department of General Surgery, University of Florida College of Medicine, 32610, Gainesville, FL, USA

    Juan Carlos Cendan MD, Edward M. Copeland III MD &  D. Scott Lind MD

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  1. Juan Carlos Cendan MD
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  2. Wiley W. Souba MD, ScD
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  3. Edward M. Copeland III MD
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  4. D. Scott Lind MD
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Cendan, J.C., Souba, W.W., Copeland, E.M. et al. Increasedl-arginine transport in a nitric oxide-producing metastatic colon cancer cell line. Annals of Surgical Oncology 3, 501–508 (1996). https://doi.org/10.1007/BF02305770

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