Modulation of receptor activity and cell cycling independently regulate retroviral gene transfer

Retroviral receptor expression in hepatocytes is upregulated during liver regeneration, although whether increased receptor expression could by itself improve hepatic gene transfer is unknown. To dissect mechanisms in retroviral gene transfer, we used an in vitro system based upon human Huh-7.MCAT-1.7 cells, which constitutively express the murine ecotropic retroviral receptor, MCAT-1. Analysis of MCAT-1 expression with a bioassay using Larginine uptake demonstrated that exposure to 10"M norepinephrine or IGM dexamethasone upregulated receptor activity by 1.5±0.2-fold and 2.2+0.6-fold, respectively, p<0.005. Addition of 20 ng/ml hepatocyte growth factor (HGF) increased arginine uptake by 3.8±0.8-fold, alongwith increased DNA synthesis, p<0.001, indicating a synergistic effect of HGF upon hormonal upregulation of MCAT-1 activity. When Huh-7.MCAT-1.7 cells were exposed to an ecotropic LacZ retrovirus at multiplicity of infection 2, stimulation with dexamethasone alone or norepinephrine, dexamethasone and HGF increased the number of X-gal stainable blue cells from 16%±4% in controls to 32%±3% and 60%±2% cells, respectively, p<0.05, whereas norepinephrine alone did not significantly increase gene transfer. To demonstrate whether superior gene transfer in response to HGF was due to receptor upregulation itself or to a combination of increased DNA synthesis, we inhibited cell cycling by a recombinant adenovirus expressing Mad (AdMad), which antagonizes c-Myc activity. Exposure of Huh-7.MCAT-1.7 cells to AdMad resulted in accumulation of cells in GO/G1, as well as G2/M; however, MCAT1 activity was preserved and arginine uptake increased in response to 10" M dexamethasone. When asynchronously cycling Huh-7.MCAT-1.7 cells were first infected with ecotropic LacZ virus and then treated with AdMad, gene transfer was decreased. In contrast, prior treatment of cells with AdMad followed 72 hrs later by retrovirus infection completely abolished gene transfer. CONCLUSIONS: Upregulation of cellular receptors facilitates retroviral gene transfer although simultaneous cell proliferation is critical for proviral integration and gene expression. These mechanistic insights shall facilitate strategies for hepatic gene therapy.