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Evaluation of p21CIP1/WAF1 expression and the appearance of γH2AX foci after photon based irradiation
Introduction Radiation therapy, typically in combination with chemotherapy (radiochemotherapy), is a major procedure in the treatment of head and neck squamous cell carcinoma (HNSCC). Earlier studies reported about a co-localization of the cell cycle inhibitor p21CIP1/WAF1 with γH2AX foci, which form after DNA damage, e.g. as a result of irradiation, in the nucleus of the cell. In this context, the potential use of p21CIP1/WAF1 as a sensor of DNA damage was discussed. In the present study, we evaluated the formation of photon irradiation-induced formation of γH2AX foci as well as expression of p21CIP1/WAF1.
Materials and methods Photon irradiation was performed with 1, 2, 4 or 8 Gy, followed by incubation of cells for 1, 2, 4, 8, 12 or 24h under standard culture conditions. Cell lines (UPCI:SCC154, L929) were stained with an antibody directed against phosphorylated γH2AX(ser139) and γH2AX(ser139) positive areas of DNA damage were analyzed microscopically. p21CIP1/WAF1 expression was quantified by digital image analysis of p21CIP1/WAF1 specific immunoblots.
Results Irradiated cells exhibited dose-dependent DNA damage (γH2AX Foci), which at 8 Gy remained significantly elevated after 24h. The cell cycle inhibitor p21CIP1/WAF1 was induced in a time-delayed manner relative to the appearance of γH2AX foci at all tested doses (1, 2, 4 und 8 Gy) and remained significantly elevated 24h after irradiation.
Conclusion Our observations are backing reports of other groups regarding a γH2AX-dependent induction of p21CIP1/WAF1 after irradiation and the possible benefit of p21CIP1/WAF1 as a marker of DNA damage.
10 June 2020 (online)
© Georg Thieme Verlag KG
Stuttgart · New York