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

2004 ◽  
Vol 286 (3) ◽  
pp. G467-G478 ◽  
Author(s):  
Bryan C. Fuchs ◽  
J. Christian Perez ◽  
Julie E. Suetterlin ◽  
Sofia B. Chaudhry ◽  
Barrie P. Bode

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.

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Eva-Maria Sedlmeier ◽  
Dorothy M. Meyer ◽  
Lynne Stecher ◽  
Manuela Sailer ◽  
Hannelore Daniel ◽  
...  

Abstract Background Previously, we revealed sexually dimorphic mRNA expression and responsiveness to maternal dietary supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in placentas from a defined INFAT study subpopulation. Here, we extended these analyses and explored the respective placental microRNA expression, putative microRNA-mRNA interactions, and downstream target processes as well as their associations with INFAT offspring body composition. Results We performed explorative placental microRNA profiling, predicted microRNA-mRNA interactions by bioinformatics, validated placental target microRNAs and their putative targets by RT-qPCR and western blotting, and measured amino acid levels in maternal and offspring cord blood plasma and placenta. microRNA, mRNA, protein, and amino acid levels were associated with each other and with offspring body composition from birth to 5 years of age. Forty-six differentially regulated microRNAs were found. Validations identified differential expression for microRNA-99a (miR-99a) and its predicted target genes mTOR, SLC7A5, encoding L-type amino acid transporter 1 (LAT1), and SLC6A6, encoding taurine transporter (TauT), and their prevailing significant sexually dimorphic regulation. Target mRNA levels were mostly higher in placentas from control male than from female offspring, whereas respective n-3 LCPUFA responsive target upregulation was predominantly found in female placentas, explaining the rather balanced expression levels between the sexes present only in the intervention group. LAT1 and TauT substrates tryptophan and taurine, respectively, were significantly altered in both maternal plasma at 32 weeks’ gestation and cord plasma following intervention, but not in the placenta. Several significant associations were observed for miR-99a, mTOR mRNA, SLC7A5 mRNA, and taurine and tryptophan in maternal and cord plasma with offspring body composition at birth, 1 year, 3 and 5 years of age. Conclusions Our data suggest that the analyzed targets may be part of a sexually dimorphic molecular regulatory network in the placenta, possibly modulating gene expression per se and/or counteracting n-3 LCPUFA responsive changes, and thereby stabilizing respective placental and fetal amino acid levels. Our data propose placental miR-99, SLC7A5 mRNA, and taurine and tryptophan levels in maternal and fetal plasma as potentially predictive biomarkers for offspring body composition.


2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Michelle M. Thiaville ◽  
Yuan‐Xiang Pan ◽  
Altin Gjymishka ◽  
Michael S. Kilberg

2008 ◽  
Vol 283 (16) ◽  
pp. 10848-10857 ◽  
Author(s):  
Michelle M. Thiaville ◽  
Yuan-Xiang Pan ◽  
Altin Gjymishka ◽  
Can Zhong ◽  
Randal J. Kaufman ◽  
...  

2010 ◽  
Vol 41 (3) ◽  
pp. 315-327 ◽  
Author(s):  
Jixiu Shan ◽  
Maria-Cecilia Lopez ◽  
Henry V. Baker ◽  
Michael S. Kilberg

Dietary protein malnutrition is manifested as amino acid deprivation of individual cells, which activates an amino acid response (AAR) that alters cellular functions, in part, by regulating transcriptional and posttranscriptional mechanisms. The AAR was activated in HepG2 human hepatoma cells, and the changes in mRNA content were analyzed by microarray expression profiling. The results documented that 1,507 genes were differentially regulated by P < 0.001 and by more than twofold in response to the AAR, 250 downregulated and 1,257 upregulated. The spectrum of altered genes reveals that amino acid deprivation has far-reaching implications for gene expression and cellular function. Among those cellular functions with the largest numbers of altered genes were cell growth and proliferation, cell cycle, gene expression, cell death, and development. Potential biological relationships between the differentially expressed genes were analyzed by computer software that generates gene networks. Proteins that were central to the most significant of these networks included c-myc, polycomb group proteins, transforming growth factor β1, nuclear factor (erythroid-derived 2)-like 2-related factor 2, FOS/JUN family members, and many members of the basic leucine zipper superfamily of transcription factors. Although most of these networks contained some genes that were known to be amino acid responsive, many new relationships were identified that underscored the broad impact that amino acid stress has on cellular function.


FEBS Letters ◽  
2004 ◽  
Vol 576 (3) ◽  
pp. 481-486 ◽  
Author(s):  
Dazhi Lai ◽  
Shaojie Weng ◽  
Cui'e Wang ◽  
Lianquan Qi ◽  
Changming Yu ◽  
...  

2004 ◽  
Vol 379 (1) ◽  
pp. 79-88 ◽  
Author(s):  
Van LEUNG-PINEDA ◽  
YuanXiang PAN ◽  
Hong CHEN ◽  
Michael S. KILBERG

mRNA abundance for a number of genes is increased by amino acid limitation. From an array screening study in HepG2 human hepatoma cells, it was established that one set of genes affected by amino acid availability is the set associated with cell-cycle control. The present study describes the increased expression of both mRNA and protein for the cyclin-dependent kinase inhibitors p21 and p27 in response to deprivation of HepG2 cells for a single essential amino acid, histidine. The increase in p21 and p27 mRNA content depended on de novo protein synthesis and involved a post-transcriptional mRNA stabilization component. For p21, increase in mRNA by histidine depletion appeared to be independent of p53 transactivation, and the absolute level of p53 protein was unaffected by this treatment. Histidine limitation caused an increase in the phosphorylation of ERK1/ERK2 (extracellular-signal-regulated kinase), and inhibition of the ERK signal transduction pathway resulted in a reduction in the starvation-dependent increase in p21 mRNA. Blockade of the phosphoinositide 3-kinase and mTOR (mammalian target of rapamycin) pathways also blunted the increase in p21 mRNA content. These results document the amino acid-dependent control of the synthesis of specific cell-cycle regulators and help to explain the block at G1 phase after amino acid limitation.


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