Non-invasive monitoring of therapeutic effect of siRNA-mediated radiation sensitization in human prostate cancer xenografts
Director:
Theodore DeWeese, M.D., Ph.D.
Brief Discription and findings:
The purpose of this project was to determine if non-invasive imaging techniques can provide reliable information regarding radiation sensitization induced by siRNA’s targeting the DNA damage sensors ATM and ATR. siRNAs targeting ATM and DNA-PKcs gave rise to a 2-4 fold increase in radiation sensitivity compared to untransfected and control vector-transfected cells at the clinically relevant dose range of 2-6Gy. Clinical translation of these genetic radiation-response modifiers would be facilitated by pre-clinical assessment of tumor response using non-invasive imaging methods that are already in the clinic. We experienced difficulty expressing a siRNA targeting DNA-PK and ATM in the context of an adenovirus possibly since cis-acting elements were interacting with the U6 promoter, inhibiting its expression. We therefore studied an alternative pol III promoter system, the adenoviral VA1 promoter, and have shown that our siRNA’s can be driven by this promoter in a plasmid-based system resulting in reductions in DNA-PK target protein expression equivalent to the reductions seen using the U6 promoter system. We also tested the ability of the VA1 promoter to drive expression of a siRNA targeting luciferase. We created 293 and DU 145 cells that constitutively express luciferase and serve as efficient systems for testing our siRNA-based protein knockdown systems. We created a plasmid-based siRNA targeting luciferase using the VA1 promoter and transfected both 293 luciferase-expressing and DU145 luciferase-expressing cells and observed that VA1 was capable of driving expression of a targeted siRNA resulting in substantial reductions in luciferase protein levels. However, when we incorporated the VA1 promoter into our adenoviral backbone to drive expression of our siRNA targeting DNA-PK or luciferase and transfected DU 145 cells we were unable to detect a significant decrement in either DNA-PK protein level or luciferase protein level.
[Back]
