Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo
Promoter Regions, Genetic
Ku70 is one component of a protein complex, Ku70 and Ku80, that functions as a heterodimer to bind DNA double-strand breaks and activates DNA-dependent protein kinase. Our previous study with Ku70-/- and Ku80-/- mice, and cell lines has shown that Ku70- and Ku80-deficiency compromises the ability of cells to repair DNA double-strand breaks, increases radiosensitivity of cells, and enhances radiation-induced apoptosis. In this study, we examined the feasibility of using adenovirus-mediated, heat-activated expression of antisense Ku70 RNA as a gene therapy paradigm to sensitize cells and tumors to ionizing radiation. First, we performed experiments to test the heat inducibility of heat shock protein (hsp) 70 promoter and the efficiency of adenovirus-mediated gene transfer in rodent and human cells. Replication-defective adenovirus vectors were used to introduce a recombinant DNA construct, containing the enhanced green fluorescent protein (EGFP) under the control of an inducible hsp70 promoter, into exponentially growing cells. At 24 h after infection, cells were exposed to heat treatment, and heat-induced EGFP expression at different times was determined by flow cytometry. Our data clearly show that heat shock at 42 degrees C, 43 degrees C, or 44 degrees C appears to be equally effective in activating the hsp70 promoter-driven EGFP expression (>300-fold) in various tumor cells. Second, we have generated adenovirus vectors containing antisense Ku70 under the control of an inducible hsp70 promoter. Exponentially growing cells were infected with the adenovirus vector, heat shocked 24 h later, and the radiosensitivity determined 12 h after heat shock. Our data show that heat shock induces antisense Ku70 RNA, reduces the endogenous Ku70 level, and significantly increases the radiosensitivity of the cells. Third, we have performed studies to test whether Ku70 protein level can be down-regulated in a solid mouse tumor (FSa-II), and whether this results in enhanced radiosensitivity in vivo, as assessed by in vivo/in vitro colony formation and by the tumor growth delay. Our data demonstrate that heat-shock-induced expression of antisense Ku70 RNA attenuates Ku70 protein expression in FSa-II tumors, and significantly sensitizes the FSa-II tumors to ionizing radiation. Taken together, our results suggest that adenovirus-mediated, heat-activated antisense Ku70 expression may provide a novel approach to radiosensitize human tumors.