Simultaneous Up-regulation of Viral Receptor Expression and DNA Synthesis Is Required for Increasing Efficiency of Retroviral Hepatic Gene Transfer

• Published in: The Journal of biological chemistry Vol. 273; no. 19; pp. 11954 - 11961
• Main Authors: Ott, M, Stockert, R J, Ma, Q, Gagandeep, S, Gupta, S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 08.05.1998
• Subjects: Amino Acid Transport Systems, Basic; Arginine - metabolism; Carrier Proteins - metabolism; Cell Division; DNA - biosynthesis; Gene Transfer Techniques; Genetic Vectors; Humans; Liver - virology; Lysine - metabolism; Membrane Proteins - metabolism; Receptors, Virus - metabolism; Retroviridae - genetics; Retroviridae - growth & development; Up-Regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.19.11954

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 V max 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 G 0 /G 1 , 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.