Radiation therapy is widely used in the treatment of localized breast cancer. A major focus of current radiation research is to elucidate molecular and cellular mechanisms that determine sensitivity or resistance to ionizing radiation. Treatment failure leading to disease recurrence is likely to result from the capacity of the tumor cell to effectively repair the DNA damage induced by radiation. Thus, DNA damage repair proteins are ideal targets for anti-cancer therapies because the inability to repair double strand breaks can lead to cell death. Strategies that selectively target and/or sensitize tumor cells to radiation should result in increased tumor cell death and ultimately increased patient survival. In the current work, we have developed a system for genetic disruption of DNA damage repair through targeting of XRCC4, a protein essential for Non Homologous End Joining (NHEJ), the predominant mammalian DNA damage repair pathway. Based on previous structural data for XRCC4, we created a fragment of the XRCC4 protein that will dimerize and bind to its heterodimeric partner, ligase IV, interfering with the functional activity of ligase IV. The fragment of XRCC4 DNA was inserted into a plasmid expressing the FLAG peptide and an adenovirus was constructed expressing the FLAG-tagged fragment of XRCC4. We show by western blot analysis using an antibody against the FLAG peptide and against XRCC4 that MDA-MB231 breast tumor cells express the fragment of XRCC4. The FLAG-tagged XRCC4 fragment was immunoprecipitated from both cytosolic and nuclear fractions. Probing with a ligase IV antibody demonstrated that the XRCC4 fragment bound to ligase IV in MDA-MB231 cells. We also found full length XRCC4 in these immunoprecipitates, consistent with the association of ligase IV with 2 molecules of XRCC4. Clonogenic survival analysis revealed that the expression of the XRCC4 fragment sensitized MDA-MB231 cells to ionizing radiation when compared to uninfected cells and cells infected with a control adenovirus. Sensitization by the XRCC4 fragment may be limited because of full length XRCC4 bound to ligase IV. As the XRCC4 fragment is able to bind ligase IV, it is likely that the XRCC4 fragment is able to interfere with NHEJ and cause radiosensitization. Supported in part by the DOD Breast Cancer Research Program Award DAMD17-03-1-0414, U.S. Army Medical Research and Materiel Command Award DAMD 17-01-1-0441 and NIH Grant CA40615.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]