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LIVER AND BILIARY TRACT

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

Department of Biology, Saint Louis University, St. Louis, Missouri 63103-2010

Submitted 8 August 2003 ; accepted in final form 12 October 2003

Amino acid transporter B⁰/ASC transporter 2 (ATB⁰/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 ATB⁰/ASCT2 antisense RNA expression plasmid under the transcriptional control of mifepristone, a synthetic steroid. Induced antisense RNA expression in monolayer cultures decreased ATB⁰/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 ATB⁰/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.

caspase; glutamine; hepatocellular carcinoma

This article has been cited by other articles:

				B. C. Fuchs, R. E. Finger, M. C. Onan, and B. P. Bode ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells Am J Physiol Cell Physiol, July 1, 2007; 293(1): C55 - C63. [Abstract] [Full Text] [PDF]