Radiosensitization of Oropharyngeal Squamous Cell Carcinoma Cells by Human Papillomavirus 16 Oncoprotein E6∗I

Abstract

Patients with oropharyngeal squamous cell carcinoma (OSCC) whose disease is associated with high-risk human papillomavirus (HPV) infection have a significantly better outcome than those with HPV-negative disease, but the reasons for the better outcome are not known. We postulated that they might relate to an ability of HPV proteins to confer a better response to radiotherapy, a commonly used treatment for OSCC. We stably expressed the specific splicing-derived isoforms, E6∗I and E6∗II, or the entire E6 open reading frame (E6total), which gives rise to both full length and E6∗I isoforms, in OSCC cell lines. Radiation resistance was measured in clonogenicity assays, p53 activity was measured using transfected reporter genes, and flow cytometry was used to analyze cell cycle and apoptosis. E6∗I and E6total sensitized the OSCC cells to irradiation, E6∗I giving the greatest degree of radiosensitization (approximately eightfold lower surviving cell fraction at 10 Gy), whereas E6∗II had no effect. In contrast to radiosensitivity, E6∗I was a weaker inhibitor than E6total of tumor suppressor p53 transactivator activity in the same cells. Flow cytometric analyses showed that irradiated E6∗I expressing cells had a much higher G2M:G1 ratio than control cells, indicating that, after G2, cells were diverted from the cell cycle to programmed cell death. This study supports a role for E6∗I in the enhanced responsiveness of HPV-positive oropharyngeal carcinomas to p53-independent radiation-induced death.