Dna double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity

Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs), DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. Methods and Materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE), DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham), DNA ligase activity in crude cell extracts was measured using [5'-P-33] Poly (dA).(Oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e(-alpha D-beta D2), the two cell lines roughly have the same alpha value (similar to 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2) +/- 0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2) +/- 0.004 Gy(-2) [SEM]), Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE, The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A (p < 0.05), The constitutive level of DNA ligase activity was similar in the two cell lines. Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity, (C) 1998 Elsevier Science Inc.