Zhang Mei, Hong Angela M
Faculty of Medicine and Health, Central Clinical School, The University of Sydney, NSW, Australia.
Department of Radiation Oncology, Chris O'Brien Lifehouse, Camperdown, NSW, Australia.
Oncotarget. 2020 Apr 21;11(16):1417-1426. doi: 10.18632/oncotarget.27535.
Patients with Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) has better outcomes than those with HPV-negative OPSCC. This may be related to its enhanced radiosensitivity. This study examined the effect of HPV and its E6 oncoprotein on the morphology, radiosensitivity, and repair of radiation-induced DNA damage. HPV-negative UM-SCC4 with and without transfection of HPV E6 oncoprotein, HPV-negative UPCI-SCC-089 and HPV-positive UPCI-SCC-099 cell lines were used in this study. The radiosensitivity and morphological changes after radiation were determined by clonogenic assay. Radiation-induced double-strand breaks in the DNA was measured by γ-H2AX foci immunofluorescent assay. The survival fraction after 10 Gy was significantly lower for the HPV-positive SCC-099 cells than for the HPV-negative cells ( = 0.03). The levels of γ-H2AX foci formation and retention were time and cell line-dependent. The γ-H2AX level started to increase at 1 hour and peaked at 4 hours after 10 Gy radiation in the HPV-negative SCC-089 and UM-SCC4 cells before reducing to negligible level ( = 0.0001). In contrast, the HPV-positive UPCI-SCC-099 cells displayed persistent γ-H2AX activity; the expression of γ-H2AX remained high at 48 hours post radiation ( = 0.001). Transfection with the E6 oncoprotein prolonged γ-H2AX formation up to 24 hours in HPV-negative SCC4 cells. HPV-positive SCC-099 cells were more likely to show the classical apoptotic changes of increased cell thickness and increased motility after radiation. This study confirmed that HPV-positive OPSCC was more radiosensitive. Transfection with the E6 oncoprotein enhanced the radiosensitivity in HPV-negative OPSCC by impairing the DNA repair mechanism and enhancing apoptotic cell death.
人乳头瘤病毒（HPV）阳性的口咽鳞状细胞癌（OPSCC）患者比HPV阴性的OPSCC患者预后更好。这可能与其增强的放射敏感性有关。本研究检测了HPV及其E6癌蛋白对辐射诱导的DNA损伤的形态、放射敏感性和修复的影响。本研究使用了转染和未转染HPV E6癌蛋白的HPV阴性UM-SCC4细胞系、HPV阴性UPCI-SCC-089细胞系和HPV阳性UPCI-SCC-099细胞系。通过克隆形成试验测定辐射后的放射敏感性和形态变化。通过γ-H2AX灶免疫荧光试验测量DNA中辐射诱导的双链断裂。10 Gy辐射后，HPV阳性的SCC-099细胞的存活分数显著低于HPV阴性细胞（P = 0.03）。γ-H2AX灶形成和保留水平与时间和细胞系有关。在10 Gy辐射后，HPV阴性的SCC-089和UM-SCC4细胞中，γ-H2AX水平在1小时开始升高，4小时达到峰值，然后降至可忽略不计的水平（P = 0.0001）。相比之下，HPV阳性的UPCI-SCC-099细胞显示出持续的γ-H2AX活性；辐射后48小时，γ-H2AX的表达仍然很高（P = 0.001）。用E6癌蛋白转染可使HPV阴性的SCC4细胞中γ-H2AX形成延长至24小时。HPV阳性的SCC-099细胞在辐射后更有可能表现出细胞厚度增加和运动性增加的典型凋亡变化。本研究证实HPV阳性的OPSCC对辐射更敏感。用E6癌蛋白转染通过损害DNA修复机制和增强凋亡性细胞死亡来增强HPV阴性OPSCC的放射敏感性。
