Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis

Abstract

Background: 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 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.

Results: Initial 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.

Conclusion: Arginine-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.

Figure 1

Comparison of arginine-responsive gene sets in normal and tumor cells. (A) a: Arginine-responsive genes in Normal Rat Hepatic Cells; b: Arginine-responsive genes in both Normal and Tumor Rat Hepatic Cells; c: Arginine-responsive genes in Tumor Rat Hepatic Cells. (B) a: Genes with expression Increase in Normal b: Genes with expression Increase in both Normal and Tumor; c: Genes with expression Increase in Tumor. (C) a: Genes with expression Decrease in Normal; b: Genes with expression Decrease in both Normal and Tumor; c: Genes with expression Decrease in Tumor.

Figure 2

Northern blot confirmation of selected rat hepatic gene expression in normal and tumor cells in response to 18 hr arginine deprivation. Total RNA (10 ug per culture condition) was electrophoresed and blotted to nylon membranes for sequential hybridization with P-32 labeled cDNA probes for p21, GADD45, Insig-1, 4F2, LAT1, LAT2 and GAPDH. Autoradiographic exposures shown represent maximal differences observed between + and - arginine conditions. The ethidium bromide stained blot prior to hybridization is shown below for comparison of loading between lanes and evidence of intact RNA quality.

Figure 3

RT-PCR confirmation of selected rat hepatic gene expression in normal and tumor cells in response to 18 hr arginine deprivation. Semi-quantitative Reverse Transcription Polymerase Chain Reaction was used to assess relative expression of rat LDLr, farnesyl diphosphate synthase (FDPS), GADD45, Insig-1, and GAPDH for cells cultured with (+) or without (-) arginine, Adult liver served as a positive control. DNase treated total RNA was reverse-transcribed with and without (negative control) reverse transcriptase. A. Electrophoresis of PCR products was performed in 2% agarose, 1× TAE gel and visualized by ethidium bromide staining; 1 kb DNA marker was used to verify size of the PCR products. B. Densitometry of gel bands was assessed via LabWorks Software and values were ratioed to GAPDH to provide relative comparisons between Arginine + and Arginine - conditions.