PARENT SESSION
1:30 PM to 2:30 PM
Monday, April 22, 2002
Poster Session 4 Gene Therapy
Room: Nevada 4-5
(MP04-39) Adenovirus-mediated antisense Ku70 gene transfer increases the radiosensitivity of human malignant gliomas.
Fuqiu, He1, Shao, Rachel1, Urano, Muneyasu1, Xu, Ming1, Julien, Terrance1, Borrelli, Mike2, Gutin, Philip1, Li, Gloria*,1, 1 Memorial Sloan-Kettering Cancer Center, New York, NY2 William Beaumont Hospital, Royal Oak, MI
ABSTRACT-
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. Generation of Ku70-/- and Ku80-/- mice and cell lines has confirmed that Ku70 and Ku80 play an essential role in DNA damage repair. Specifically, Ku70- and Ku80-deficiency compromises cells' ability to repair DNA double-strand breaks, increases cells' radiosensitivity and enhances radiation-induced apoptosis. Because of the extreme radioresistance of glioblastoma cells, we examined the feasibility of using adenovirus (ad)-mediated, heat-activated expression of antisense Ku70 RNA as a gene therapy paradigm to sensitize glioma cells to ionizing radiation. First, we examined the heat inducibility of hsp70 promoter and the efficiency of ad-mediated gene transfer in human glioblastoma U-87 MG cells. Replication-defective ad vectors were used to introduce a recombinant DNA construct, containing EGFP under the control of a hsp70 promoter, into U-87 MG cells. The heat-induced EGFP expression at different times after heat shock was determined by flow cytometry. Our data clearly show that EGFP expression can be activated to greater than several hundred fold. Second, we generated ad vectors containing antisense Ku70 under the control of an inducible hsp70 promoter. Exponentially growing U-87 MG cells were infected with the ad vector, heat shocked 24 hr later, and the cells' radiosensitivity determined. Our data show that heat shock induced antisense Ku70 RNA expression, reduced the endogenous Ku70 level, and significantly increased the radiosensitivity of U-87 MG cells. Third, we performed studies to test whether Ku70 protein level can be down-regulated in solid mouse tumor (FSa-II), and whether this results in enhanced radiosensitivity in vivo. Our data showed that heat-shock-induced expression of antisense Ku70 RNA attenuated Ku70 level, and significantly sensitized the FSa-II tumors to ionizing radiation as determined by in vivo/in vitro colony formation assay and by the tumor growth delay endpoint. Taken together, our results support that ad-mediated, heat-activated antisense-Ku70 expression may provide a novel approach to radiosensitize human malignant glioma cells.
KEYWORDS: gene therapy, Ku protein, radiation sensitivity, heat shock