Inducible antisense RNA targeting amino acid transporter ATB0/ASCT2 elicits apoptosis in human hepatoma cells.

Amino acid transporter B(0)/ASC transporter 2 (ATB(0)/ASCT2) is responsible for most glutamine uptake in human hepatoma cells. Because this transporter is not expressed in normal hepatocytes, we hypothesized that its expression is necessary for growth of human liver cancer cells. To test this hypothesis, Sloan Kettering hepatoma (SK-Hep) cells were stably transfected with an inducible 1.3-kb ATB(0)/ASCT2 antisense RNA expression plasmid under the transcriptional control of mifepristone, a synthetic steroid. Induced antisense RNA expression in monolayer cultures decreased ATB(0)/ASCT2 mRNA levels by 73% and glutamine transport rates by 65% compared with controls after 24 h, leading to a 98% decrease in cell number after 48 h. Cellular death was attributable to apoptosis based on cellular blebbing, caspase-3 activation, vital dye and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and poly-(ADP-ribose) polymerase (PARP) cleavage. Transporter knockdown also markedly increased activities of caspases-2 and -9, marginally enhanced caspase-8 activity, and dramatically increased ASCT1 mRNA levels, presumably as a futile compensatory response. Apoptosis elicited via transporter silencing was not attributable to the double-stranded RNA-dependent protein kinase R (PKR) pathway. For comparison, glutamine deprivation also caused apoptotic cell death but with slower temporal kinetics, stimulated caspases-2 and -3 but not caspases-8 or -9 activities, and led to considerable PARP cleavage. Thus ASCT2 suppression exerts proapoptotic effects transcending those of glutamine starvation alone. We conclude that ATB(0)/ASCT2 expression is necessary for SK-Hep cell growth and viability and suggest that it be further explored as a selective target for human hepatocellular carcinoma.

Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells.

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter B(0) (ATB(0)), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by ATB(0). The 2.9-kb ATB(0) mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. ATB(0) mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of ATB(0)-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels.