| SNAT4 (s&barbelow;odium-dependent n&barbelow;eutral a&barbelow;mino acid t&barbelow;ransporter 4), isolated in 2001, is a member of the System A and System N family of amino acid transporters. Among the SNAT family members, SNAT4 is unique in mediating cationic (essential amino acids arginine and lysine) as well as neutral amino acid uptake. SNAT4 is upregulated at the mRNA level by over an order of magnitude in response to the three-dimensional growth of HepG2 multicellular tumor spheroids in vitro, after the aggregates show signs of central necrosis (due to lack of angiogenic oxygen and nutrient supply). This observation suggested that it could play an essential role in the initial avascu1ar growth or survival of tumors. The hypothesis that SNAT4 upregulation was due cell stress caused by the microenvironments such as hypoxia or nutrient limitation known to develop during spheroid growth was tested by introduction of chemical cell-stressors (cobalt chloride and buthionine sulfoximine), oxygen deprivation, glucose limitation and amino acid starvation, alone or in combination, in cell culture media: none of these factors resulted in significant induction of SNAT4 mRNA expression. Fluorescent in situ hybridization studies suggested that SNAT4 mRNA localization patterns differed between HepG2 and Huh7 large spheroids, two hepatoma cell lines that exhibit different levels of liver-specific gene expression. Hypothesizing that SNAT4 expression could be linked to levels of differentiation, RT-PCR studies were conducted to determine the expression levels of liver-enriched transcription factors; however, no differential expression was observed between the cell lines tested, or between HepG2 cells in monolayer culture or large spheroids. However, evidence of a correlation between SNAT4 expression and the expression of the urea cycle enzyme arginase I was elucidated. These results demonstrate that SNAT4 expression: (1) is not stress-related, (2) may be linked to a differentiation program tied to urea cycle enzyme expression, and (3) may function to deliver arginine to liver cells that express arginase I. |
