Use of the human MDR1 promoter for heat-inducible expression of therapeutic genes

Abstract

The promoter of the human multidrug resistance gene (mdr1) harbors stress-responsive elements, which can be induced e.g., by heat or cytostatic drugs. In previous studies the drug-responsiveness of the mdr1 promoter was successfully used for the drug-inducible expression of the human TNF-alpha gene in vitro and in vivo. Beside the drug-responsive elements of the mdr1 promoter, heat-shock responsive elements have also been identified, which could be exploited for construction of heat-inducible expression vectors. To analyze the hyperthermia-inducibility of the mdr1 promoter we used the pmdr-p-CAT and pM3mdr-p-hTNF vector constructs. Both constructs carry the mdr1 promoter fragment spanning from -207 to +153 to drive expression of the CAT-reporter or TNF-alpha gene. We tested the heat-induced CAT-reporter and TNF-alpha expression in vitro in transduced HCT15 and HCT116 human colon carcinoma cells. For the studies the transduced tumor cells were treated with hyperthermia at 41.5 degrees C or 43 degrees C for 2 hr to induce CAT or TNF-alpha expression. Cells and supernatants were harvested before hyperthermia and at certain time points (0-120 hr) after heat shock. The heat-induced CAT-reporter expression or TNF-alpha secretion was determined by specific ELISA. The experiments indicate that hyperthermia activates the mdr1 promoter in a temperature and time dependent manner. This induction leads to an 2- to 4-fold increase in CAT-reporter or 2- to 7-fold increase in TNF alpha expression in the tumor cell lines. These experiments reveal that the mdr1 promoter driven expression of therapeutic genes can be employed for combined cancer gene therapy approaches.