Abstract

BackgroundWe have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5) in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We hypothesized that liver cell gene expression is highly sensitive to alterations in the amino acid microenvironment and that tumor cells may differ substantially in gene sets sensitive to amino acid availability. To assess the potential number and classes of hepatic genes sensitive to arginine availability at the RNA level and compare these between normal and tumor cells, we used an Affymetrix microarray approach, a paired in vitro model of normal rat hepatic cells and a tumorigenic derivative with triplicate independent replicates. Cells were exposed to arginine-deficient or control conditions for 18 hours in medium formulated to maintain differentiated function.ResultsInitial two-way analysis with a p-value of 0.05 identified 1419 genes in normal cells versus 2175 in tumor cells whose expression was altered in arginine-deficient conditions relative to controls, representing 9–14% of the rat genome. More stringent bioinformatic analysis with 9-way comparisons and a minimum of 2-fold variation narrowed this set to 56 arginine-responsive genes in normal liver cells and 162 in tumor cells. Approximately half the arginine-responsive genes in normal cells overlap with those in tumor cells. Of these, the majority was increased in expression and included multiple growth, survival, and stress-related genes. GADD45, TA1/LAT1, and caspases 11 and 12 were among this group. Previously known amino acid regulated genes were among the pool in both cell types. Available cDNA probes allowed independent validation of microarray data for multiple genes. Among genes downregulated under arginine-deficient conditions were multiple genes involved in cholesterol and fatty acid metabolism. Expression of low-density lipoprotein receptor was decreased in both normal and tumor cells.ConclusionArginine-sensitive regulation appears to be an important homeostatic mechanism to coordinate cell response and nutrient availability in hepatic cells. Genes predicted as arginine-responsive in stringent microarray data analysis were confirmed by Northern blot and RT-PCR. Although the profile of arginine-responsive genes is altered and increased, a considerable portion of the "arginome" is maintained upon neoplastic transformation.

Highlights

  • We have reported arginine-sensitive regulation of L-type amino acid transporter (LAT1) amino acid transporter (SLC 7A5) in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells

  • We previously reported that levels of the tumor-associated glycoprotein amino acid transporter, TA1/LAT1/CD98 light chain/SLC7A5, increase in normal hepatic cells under low arginine conditions, while levels are constitutive and high in hepatic tumor cells [4,5]

  • The goals of the study were to: 1) assess the scope of arginine-responsive hepatic gene expression using a welldefined in vitro rat model of normal and tumorigenic cells, 2) determine to what extent amino acid responsive regulation is retained upon transformation, and 3) provide a microarray dataset predicting novel genes and pathways subject to amino acid regulation

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Summary

Introduction

We have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5) in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We previously reported that levels of the tumor-associated glycoprotein amino acid transporter, TA1/LAT1/CD98 light chain/SLC7A5, increase in normal hepatic cells under low arginine conditions, while levels are constitutive and high in hepatic tumor cells [4,5]. Upregulation of this gene is associated with multiple cancer types and we and others have hypothesized that increased expression may provide an adaptive advantage in the tumor microenvironment where nutrients are limiting [6,7]. The goals of the study were to: 1) assess the scope of arginine-responsive hepatic gene expression using a welldefined in vitro rat model of normal and tumorigenic cells, 2) determine to what extent amino acid responsive regulation is retained upon transformation, and 3) provide a microarray dataset predicting novel genes and pathways subject to amino acid (arginine) regulation

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