Hypoxia-inducible transgene expression in differentiated human NT2N neurons--a cell culture model for gene therapy of postischemic neuronal loss.

Expression of anti-apoptotic or neurotrophic transgene proteins in hypoxic neurons may provide a novel therapeutic strategy for neuroprotection and alleviation of damage to ischemic brain areas. NT2, a human neoplastic cell line which terminally differentiates into postmitotic neurons (NT2N) by treatment with retinoic acid was used in this study as a cell culture model for human neuronal cells. The hypoxia-inducible VEGF promoter in plasmid vectors was employed to drive the expression of marker genes (luciferase) and therapeutic genes (bcl2) in hypoxic NT2 cells and NT2N neurons in culture. Cationic liposomes complexed with plasmid DNA were used for transfection of vectors with the constitutive cytomegalovirus promoter (pCMV) or the hypoxia-inducible VEGF promoter (pHRE). Hypoxic or normoxic control NT2 cells transfected with pCMV-luciferase showed high transgene expression (2.4 x 10(8) relative light units (RLU)/mg protein). Control NT2 cells transfected with pHRE-luciferase had a rather low activity (4.9 x 10(6) RLU/mg protein), which was induced 34-fold under hypoxic conditions. Four-fold induction of luciferase expression was obtained in hypoxic NT2N neurons transfected with pHRE compared with normoxic controls. The hypoxia-induced luciferase expression in NT2N cells was 34% of the activity of pCMV-luciferase under the same conditions. Transfection of NT2N neurons with pCMV-bcl2 or pHRE-bcl2 was demonstrated to reduce significantly the numbers of apoptotic cells after hypoxia. These results demonstrate efficient VEGF promoter-mediated induction of transgene expression in hypoxic human neurons. This cell culture model may be employed for the further investigation of therapeutic proteins against ischemic brain damage due to neuronal loss.