Annals of Surgical Oncology

, Volume 2, Issue 3, pp 257–265

Characterization and growth factor stimulation ofl-arginine transport in a human colon cancer cell line

  • Juan Carlos Cendan
  • Wiley W. Souba
  • Edward M. CopelandIII
  • D. Scott Lind
Original Articles

Abstract

Background: Epidermal growth factor (EGF) and transforming growth factor α (TGFα) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation.

Methods: The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [3H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [3H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGFα, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation.

Results: The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65–70%), whereas the remainder was sodium-dependent (28–30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na+-independent transporter with a Km=55.8±5.8 µM and a Vmax =710.6±87.3 pM/mg protein/30 s. Na+-independent arginine uptake was pH-insensitive and markedly inhibited by system y+ substrates L-homoarginine, L-lysine, and L-ornithine. A single Na+-dependent transporter with a Km=19.8±2.3 µM and a Vmax=159.1±8.9 pM/mg protein/30 s was identified. Na+-dependent arginine uptake was inhibited by system BO,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na+-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGFα had no effect on Na+-independent L-arginine uptake; however, Na+-dependent uptake was enhanced 60% by EGF (10 ng/ml, p<0.05) and 100% by TGFα (10 ng/ml, p<0.05), whereas cellular proliferation was increased 27% by EGF (10 ng/ml, p<0.05) and 37% by TGFα (10 ng/ml, p<0.01).

Conclusions: L-arginine transport in the SW480 colon cancer cell line is principally mediated by the Na+-independent system y+ and to a lesser extent by the Na+-dependent system BO,+. Furthermore, EGF and TGFα preferentially stimulate L-arginine uptake via the Na+-dependent transporter, ostensibly to accommodate for the mitogenic stimulus.

Key Words

Arginine Membrane transport Epidermal growth factor Transforming growth factor α 

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Copyright information

© The Society of Surgical Oncology, Inc 1995

Authors and Affiliations

  • Juan Carlos Cendan
    • 2
  • Wiley W. Souba
    • 1
  • Edward M. CopelandIII
    • 2
  • D. Scott Lind
    • 2
  1. 1.Division of Surgical Oncology, Department of SurgeryMassachusetts General Hospital, Harvard Medical SchoolBostonUSA
  2. 2.Department of General SurgeryUniversity of Florida College of MedicineGainesvilleUSA

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