title: Simultaneous up-regulation of viral receptor expression and DNA synthesis is required for increasing efficiency of retroviral hepatic gene transfer.
abstract: To understand the relative contribution of viral receptor expression and cell proliferation in retroviral gene transfer, we created human hepatocyte-derived HuH-7.MCAT-1 cell lines. These cells constitutively express the murine ecotropic retroviral receptor MCAT-1 without changes in morphology or proliferation states. The MCAT-1 receptor is also a cationic amino acid transporter, and the HuH-7.MCAT-1.7 cells showed increased Vmax of uptake and steady-state accumulation of the cationic amino acids L-arginine and L-lysine. In HuH-7.MCAT-1 cells, L-arginine uptake was significantly up-regulated by norepinephrine and dexamethasone, and hepatocyte growth factor also increased L-arginine uptake along with cellular DNA synthesis. Gene transfer was also markedly increased in HuH-7. MCAT-1.7 cells incubated with an ecotropic LacZ retrovirus, and this further increased with hormones and hepatocyte growth factor. To define whether viral receptor up-regulation by itself increased gene transfer, cell cycling was inhibited by a recombinant adenovirus expressing the Mad transcription factor (AdMad), which is a dominant-negative c-Myc regulator. This restricted cells in G0/G1, without attenuating MCAT-1 activity, as shown by flow cytometry and L-arginine uptake analysis, respectively. When asynchronously cycling HuH-7.MCAT-1.7 cells were first infected with the AdMad virus and then exposed to the ecotropic LacZ virus, gene transfer was virtually abolished. The data indicate that while up-regulation of viral receptors can greatly enhance retrovirally mediated gene transfer, DNA synthesis remains an absolute requirement for hepatic gene therapy with this approach.
authors.affiliation: Marion Bessin Liver Research Center, Gastroenterology Division, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: M
authors.initials: M
authors.lastname: Ott
authors.forename: R J
authors.initials: RJ
authors.lastname: Stockert
authors.forename: Q
authors.initials: Q
authors.lastname: Ma
authors.forename: S
authors.initials: S
authors.lastname: Gagandeep
authors.forename: S
authors.initials: S
authors.lastname: Gupta
mesh_terms: D026923:Amino Acid Transport Systems, Basic; D001120:Arginine / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division; D004247:DNA / Q000096:biosynthesis*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000821:virology*; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics* / Q000254:growth & development; D015854:Up-Regulation
chemical_list: D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008565:Membrane Proteins; D011991:Receptors, Virus; D004247:DNA; D001120:Arginine; D008239:Lysine