Effect of 4‐Nitroquinoline‐1‐Oxide on DNA damage and repair in head and neck squamous cell carcinoma cells

Abstract

Background Tobacco exposure and human papillomavirus (HPV) infection are among the main factors for development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that HPV positive (+) smokers and non-smokers have similar mutational profiles, which suggests that mutations induced in smokers could be associated with the protective effects of HPV. Our preliminary data showed that 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure induces oxidative DNA damage and DSB formation in HNSCC cells at 6 hrs. HPV+ HNSCC cells also showed a relative increase in activation of homologous recombination (HR) DNA repair proteins compared to HPV negative (-) HNSCC cells at 6 hrs. In this study, we wanted to investigate the effect of 4NQO on DNA damage and repair activation at 24 hrs in HPV+ and HPV- HNSCC cells. Methods NOK E6.E7 +/-, UPCI-SCC40 (HPV-), UM-SCC104 and UM-SCC47 (HPV+) cells were utilized to check effects of 4NQO on DNA repair. Oxidative DNA damage, double strand break formation (DSBs) and protein expression were analyzed by FPG-modified alkaline comet assay, γH2AX staining and Western analysis following treatment with 4NQO for 6 hr, replacement with fresh media, and sample collection at 24 hr. Results 4NQO increased oxidative DNA damage, single strand breaks, and yH2AX foci at 6 hrs; however, these lesions were no longer present at 24 hrs. This suggests DNA repair had probably occurred upon removal of 4NQO. We no longer observed an increase in phosphorylation of BRCA1, Rad51, H2AX, and p21 levels in HNSCC cells at 24 hr although it was present at 6 hrs. Conclusion Our in-vitro data suggests that HPV may have a role in the activation of DNA repair proteins by 4NQO at 6 hrs. This may have allowed for successful repair of DNA damage in both HPV+ and HPV- HNSCC cells by 24 hrs. Taken together, these data suggest that HPV may protect against smoking-induced mutations by preferentially increasing HR DNA repair protein activation.