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J Biol Chem, Vol. 273, Issue 19, 11954-11961, May 8, 1998

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

Michael Ott, Richard J. Stockert^§, Qiangzhong Ma, Singh Gagandeep, and Sanjeev Gupta

From the Marion Bessin Liver Research Center and the Departments of  Medicine and ^§ Biochemistry, Gastroenterology Division, Albert Einstein College of Medicine, Bronx, New York 10461

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 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.

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