1062. Potential Utility of Heat Shock Protein 70 Promoter (HSP70-P) as a Stress Responsive Promoter in Gene Delivery: HSP70-P Regulated Expression of Transgenes SOD1 and GPX1

Abstract

Cells respond to heat stress by synthesis and accumulation of several members of functionally distinct family of heat shock (or stress) proteins (HSP27, HSP60, HSP70 and HSP90). HSPs confer protection against a large array of cytotoxic reagents and are implicated in thermo tolerance development. Heat shock protein70-promoter (HSP70-P) is a stress responsive promoter, which is highly responsive to a variety of stresses, including heat stress. We hypothesized that HSP70-P or other stress-responsive promoter-driven antioxidant expression could provide reasonable sensitivity of transgene expression to heat and other stresses, while avoiding constitutive overexpression of anti-oxidant enzymes. Our eventual goal is to evaluate protection of neurons from oxidant injury such as is elicited by exposure to HIV-1 gp120. In the present study we tested HSP70-P for gene delivery. We first performed a series of transfection studies using luciferase as a reporter gene. Expression of luciferase driven by HSP70-P was triggered maximally at 8Hrs post heat shock at 410C when tested in 293 and NT-2 derived neurons. Having established the likelihood that the HSP70-P could be used as a stress-responsive promoter to drive transgene expression, we then used it to test expression of antioxidant transgenes SOD1 and GPX1 in response to heat stress. HSP-70 promoter driven expression of SOD1 and GPX1 was studied in 293 cells and in terminally differentiated NT-2 derived neuronal cells by heat stress over a range of 0-72Hr. Cells, when stressed for an optimal 4-8Hr period, induced maximal expression of these transgenes. The increase in expression was about 4-fold at the optimal time period with no further increase in expression at later time intervals. These studies using a stress responsive promoter may help to better understand the ability of heat-shock promoter to regulate expression of enzymes needed to provide anti-oxidant induce protection.