IADR Abstract Archives
Calprotectin (S100A8/A9) Regulates the DNA Damage Repair Pathway in HNSCC
Objectives: DNA damage responses (DDR) are coordinated by the ATM-Chk1/Chk2 signaling pathways and lead to cell cycle arrest and DNA repair. DNA damage repair is primarily mediated through phosphorylation of γH2AX, 53BP1, BRCA1 and SMC1. Calprotectin, a heterodimeric complex of the calcium-binding proteins S100A8 and S100A9, appears to regulate cell cycle progression, cell growth and survival. Down-regulated in head and neck squamous cell carcinoma (HNSCC), S100A8/A9 over-expression in vitro suppresses tumorigenesis by activating the G2/M checkpoint. We aimed to investigate whether S100A8/A9 participates in DNA damage repair.
Methods: S100A8/A9-expressing TR146 HNSCC cells and S100A8/A9-negative KB carcinoma cells were compared in vitro. shRNA transfections were performed to silence S100A8/A9 in TR146 cells (TR146-S100A8/A9-shRNA). A negative control cell line (TR146-shRNA-control) was also produced. KB cells were co-transfected to express either WT S100A8/A9 or S100A8/A9Δ113-114. A sham-control transfectant, KB-EGFP, was also generated. To induce double-strand breaks, cells were treated with camptothecin. γH2AX, 53BP1 and SMC1 expression was assessed using confocal immunofluorescence microscopy and western blotting.
Results: TR146-S100A8/A9-shRNA cells showed significantly less 53BP1 and γH2AX than TR146 and TR146-shRNA-control cells. After incubation with camptothecin, levels of 53BP1 and γH2AX increased in TR146 and TR146-shRNA-control cells, but were not restored in TR146-S100A8/A9-shRNA cells. As expected, 53BP1 and γH2AX expression was greater in untreated KB-S100A8/A9 cells than KB and KB-EGFP cells. Interestingly, KB and KB-S100A8/A9Δ113-114 cells (missing the singular phosphorylatable site Thr113 of S100A9) showed similar 53BP1 and γH2AX protein levels. After camptothecin treatment, KB-S100A8/A9 cells showed the highest upregulation of 53BP1 and γH2AX. SMC1 levels were not affected by the calprotectin status.
Conclusions: Reduced S100A8/A9 in HNSCC is strongly suggested to show loss of function of the DDR pathway. When expressed, calprotectin may facilitate DNA repair mechanisms, cellular response to genotoxic agents, and genome integrity.
Methods: S100A8/A9-expressing TR146 HNSCC cells and S100A8/A9-negative KB carcinoma cells were compared in vitro. shRNA transfections were performed to silence S100A8/A9 in TR146 cells (TR146-S100A8/A9-shRNA). A negative control cell line (TR146-shRNA-control) was also produced. KB cells were co-transfected to express either WT S100A8/A9 or S100A8/A9Δ113-114. A sham-control transfectant, KB-EGFP, was also generated. To induce double-strand breaks, cells were treated with camptothecin. γH2AX, 53BP1 and SMC1 expression was assessed using confocal immunofluorescence microscopy and western blotting.
Results: TR146-S100A8/A9-shRNA cells showed significantly less 53BP1 and γH2AX than TR146 and TR146-shRNA-control cells. After incubation with camptothecin, levels of 53BP1 and γH2AX increased in TR146 and TR146-shRNA-control cells, but were not restored in TR146-S100A8/A9-shRNA cells. As expected, 53BP1 and γH2AX expression was greater in untreated KB-S100A8/A9 cells than KB and KB-EGFP cells. Interestingly, KB and KB-S100A8/A9Δ113-114 cells (missing the singular phosphorylatable site Thr113 of S100A9) showed similar 53BP1 and γH2AX protein levels. After camptothecin treatment, KB-S100A8/A9 cells showed the highest upregulation of 53BP1 and γH2AX. SMC1 levels were not affected by the calprotectin status.
Conclusions: Reduced S100A8/A9 in HNSCC is strongly suggested to show loss of function of the DDR pathway. When expressed, calprotectin may facilitate DNA repair mechanisms, cellular response to genotoxic agents, and genome integrity.