IADR Abstract Archives
Hypoxia Induces DNA-Repair Protein DDB2 in Oral Cancer
Objectives: Objective: Hypoxia-induced drug resistance is a major obstacle of effective cancer therapy. Previous studies show that hypoxia plays a major role in promoting invasiveness and metastasis of cancer cells. DDB2 is a sensor of DNA damage and binds to a broad range of damaged-DNA to initiate the Nuclear Excision Repair (NER) process. Our studies show that besides its role in NER, DDB2 inhibits EMT and metastasis process of cancer progression. We also show that loss of DDB2 imparts drug resistance to tumor cells by blocking ROS accumulation and apoptosis. Metastatic oral cancer cells express significantly lower level of DDB2. In this study, we analyzed the effect of low-oxygen stress of hypoxic growth on DDB2 expression in oral cancer cells.
Methods: Methods: Cell culture: oral SCC (squamous cell carcinoma) cells, SCC-09 and SCC-15 were cultured in DMEM containing 10% FBS. HOK-TERT (telomerase-immortalized) oral keratinocyte cells were grown in the synthetic medium, KBM-Gold from Lonza. Hypoxic growth: for hypoxia treatment, cells were incubated in hypoxia chamber with 1% O2. Western Blot: cell extracts were made in RIPA buffer, separated by SDS-PAGE and analyzed using Western Blot.
Results: Results: Four well characterized oral SCC cell lines, SCC4, SCC9, SCC15, SCC25 express significantly low level of DDB2 in comparison to HOK-TERT immortalized oral epithelial cell line. HOK-TERT, SCC-09 and SCC-15 cells were grown in normoxia (20% oxygen) and hypoxia (1% oxygen) for 24h, 48h, and 72h. Expression of the hypoxia-inducible factor, HIF-1α was used to validate induction of hypoxia. Our results show significant upregulation of DDB2 in normal immortalized HOK-TERT cells (p53-wild type) and SCC9 (p53-null) and SCC15 (p53-mutant) oral SCC cells.
Conclusions: Conclusion: We show that the expression of DDB2 is significantly upregulated in immortalized oral keratinocytes and in oral SCC cell lines. These results suggest a regulatory role of DDB2 in hypoxic tumors.
Methods: Methods: Cell culture: oral SCC (squamous cell carcinoma) cells, SCC-09 and SCC-15 were cultured in DMEM containing 10% FBS. HOK-TERT (telomerase-immortalized) oral keratinocyte cells were grown in the synthetic medium, KBM-Gold from Lonza. Hypoxic growth: for hypoxia treatment, cells were incubated in hypoxia chamber with 1% O2. Western Blot: cell extracts were made in RIPA buffer, separated by SDS-PAGE and analyzed using Western Blot.
Results: Results: Four well characterized oral SCC cell lines, SCC4, SCC9, SCC15, SCC25 express significantly low level of DDB2 in comparison to HOK-TERT immortalized oral epithelial cell line. HOK-TERT, SCC-09 and SCC-15 cells were grown in normoxia (20% oxygen) and hypoxia (1% oxygen) for 24h, 48h, and 72h. Expression of the hypoxia-inducible factor, HIF-1α was used to validate induction of hypoxia. Our results show significant upregulation of DDB2 in normal immortalized HOK-TERT cells (p53-wild type) and SCC9 (p53-null) and SCC15 (p53-mutant) oral SCC cells.
Conclusions: Conclusion: We show that the expression of DDB2 is significantly upregulated in immortalized oral keratinocytes and in oral SCC cell lines. These results suggest a regulatory role of DDB2 in hypoxic tumors.