Inhibition of DNA-end binding complex formation as a mechanism for celecoxib-induced radioenhancement in human head and neck carcinoma cells
Authors: Uma Raju, Hisanori Ariga, David Valdecanas, Zhen Fan, Kian K. Ang, Luka Milas
Journal: Cancer Research (2004)

1352  Introduction: Radiotherapy is a key component in the treatment of locally advanced colorectal carcinoma. Some colorectal tumors, however, respond poorly to irradiation and the exact mechanism by which these tumors fail to respond to radiotherapy remains unclear. p53 and p21 status are known to be important mediators of the cell cycle and may be involved in tumor radio-responsiveness. The goal of this study was to examine the role of p53 and p21 in the response of HCT-116 colon carcinoma cells to ionizing irradiation (IR). Methods: The colon carcinoma cell line HCT-116 and its isogenic clones (HCT-116 p53-/p21wt, HCT-116 p53wt/p21-) were utilized for these studies. Asynchronous cells were irradiated and then assessed by clonogenic assay and flow cytometry (FACS). The expression of key cell cycle regulators was measured using Western blot analysis. Results: Sixteen hours after WT cells were exposed to 6 Gy of IR a decrease in S (25% ± 3), a corresponding increase in G2 (30% ± 4), and a stable G1 population (3% ± 2) were noted. In contrast, both p53- and p21- cells showed a marked increase in G2 (62% ± 5 and 48% ± 6, respectively) with a decrease in both G1 (22% ± 3 and 15% ± 3, respectively) and S (40% ± 5 and 34% ± 4, respectively). Examination of the kinetics of the IR induced cell cycle phase changes was performed by exposing the isogenic cells to low (2 Gy) and moderate (6 Gy) IR after which the cells were harvested for FACS over a 48h period (1.5 X doubling time). Both 2 Gy and 6 Gy IR resulted in a sustained (33h) G1 block in WT cells, which began at 12h and peaked at 24h. Although 2 Gy IR induced a similar G1 arrest in p53- cells, the block was significantly attenuated and of shorter duration (12h); no changes were noted in p21- cells. Higher 6 Gy IR resulted in a sustained (18h) G2 block in p53- cells, but only an abbreviated (8h) G2 arrest in p21- cells. Consistent with cell cycle kinetics, western blot analysis of irradiated WT cells showed a 3-fold induction of p53, a 4-fold induction of p21, and a 2-fold decrease in pRb. p53- cells showed similar changes in p21 and pRb, while p21- cells failed to show significant changes in any of the cell cycle regulators examined. Survival analysis revealed that p21- cells were the most radioresistant with 20.8% cell survival. In contrast, WT cells were the most radiosensitive with only 8.9% cell survival, while p53 deficient cells displayed intermediate radiosensitivity (13.6%). Analysis of the apoptotic pathway showed upregulation of the pro-apoptotic proteins BAX and caspase 3 in WT and p53- cells, but not p21- cells. Conclusion: IR induces a differential cell cycle arrest in colon carcinoma cells depending on the IR dose and the p53/p21 status of the tumor. HCT-116 p21- cells were the most radioresistant, yet showed the least amount of cell cycle checkpoint activation after IR. Our data suggests that this radioresistance may be mediated through the inability of p21- cells to activate pro-apoptotic pathways.