No abstract

Much has been learned about plasma membrane glutamine transporter activities in health and disease over the past 30 years, including their potential regulatory role in metabolism. Since the 1960s, discrimination among individual glutamine transporters was based on functional characteristics such as substrate specificity, ion dependence, and kinetic and regulatory properties. Within the past two years, several genes encoding for proteins with these defined activities (termed "systems") have been isolated from human and rodent cDNA libraries and found to be distributed among four distinct gene families. The Na(+)-dependent glutamine transporter genes isolated thus far are System N (SN1), System A (ATA1, ATA2), System ASC/B(0) (ASCT2 or ATB(0)), System B(0,+) (ATB(0,+)) and System y(+)L (y(+)LAT1, y(+)LAT2). Na(+)-independent glutamine transporter genes encoding for System L (LAT1, LAT2) and System b(0,+) (b(0,+)AT) have also been recently isolated, and similar to y(+)L, have been shown to function as disulfide-linked heterodimers with the 4F2 heavy chain (CD98) or rBAT (related to b(0,+) amino acid transporter). In this review, the molecular features, catalytic mechanisms and tissue distributions of each are addressed. Although most of these transporters mediate the transmembrane movement of several other amino acids, their potential roles in regulating interorgan glutamine flux are discussed. Most importantly, these newly isolated transporter genes provide the long awaited tools necessary to study their molecular regulation during the catabolic states in which glutamine is considered to be "conditionally essential."

System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by approximately 50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.

Inflammatory mediators including chemokines play a critical role in acute pancreatitis. The precise nature of early inflammatory signals within the pancreas remains, however, unclear. We examined the ability of isolated pancreatic acini to synthesize CC chemokine monocyte chemotactic protein-1 (MCP-1) and CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) and the response to the secretagogue cerulein at physiological and supraphysiological concentrations. Isolated rat pancreatic acini maintained in short-term (< or =48 h) primary culture constitutively synthesized MCP-1 and CINC. Cerulein (10(-7) M; supramaximal dose) increased production of MCP-1 but not CINC. Cerulein-induced increase in MCP-1 synthesis was accompanied by increase in nuclear factor (NF)-kappaB activation shown by EMSA. Pretreatment with NF-kappaB inhibitors N-acetylcysteine (NAC) and N-tosylphenyalanine chloromethyl ketone (TPCK) blocked cerulein-induced NF-kappaB activation and abolished cerulein's effect on MCP-1 synthesis. Pretreatment with calcium antagonist BAPTA-AM also blocked cerulein's effect on MCP-1 synthesis. These results indicate that isolated acini synthesize MCP-1 and CINC and support the idea of acinar-derived chemokines as early mediators of inflammatory response in acute pancreatitis. Although cerulein hyperstimulation increased MCP-1 synthesis by a calcium-dependent mechanism involving NF-kappaB activation, CINC synthesis was not affected. This suggests that regulation of CC and CXC chemokines within acinar cells may be quite different.

Viruses used for gene therapy are usually genetically modified to deliver therapeutic transgenes and prevent viral replication. In contrast, replication-competent viruses may be used for cancer therapy because replication of some viruses within cancer cells can result in their destruction (oncolysis). Viral ribonucleotide reductase expression is defective in the HSV1 mutant hrR3. Cellular ribonucleotide reductase, which is scarce in normal liver and abundant in liver metastases, can substitute for its viral counterpart to allow hrR3 replication in infected cells. Two or three log orders more of hrR3 virions are produced from infection of colon carcinoma cells than from infection of normal hepatocytes in viral replication assays. This viral replication is oncolytic. A single intravascular administration of hrR3 into immune-competent mice bearing diffuse liver metastases dramatically reduces tumor burden. hrR3-mediated tumor inhibition is equivalent in immune-competent and immune-incompetent mice, suggesting that viral oncolysis and not the host immune response is the primary mechanism of tumor destruction. HSV1-mediated oncolysis of diffuse liver metastases is effective in mice preimmunized against HSV1. These results indicate that replication-competent HSV1 mutants hold significant promise as cancer therapeutic agents. Yoon, S. S., Nakamura, H., Carroll, N. M., Bode, B. P., Chiocca, E. A., Tanabe, K. K. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.