Katerji Meghri, Bertucci Antonella, Filippov Valery, Vazquez Marcelo, Chen Xin, Duerksen-Hughes Penelope J
Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, United States.
Department of Radiation Medicine, Loma Linda University Medical Center, Loma Linda, CA, United States.
Front Oncol. 2022 Sep 2;12:928545. doi: 10.3389/fonc.2022.928545. eCollection 2022.
High-risk human papillomaviruses (HPVs) cause virtually all cervical cancer cases and are also associated with other types of anogenital and oropharyngeal cancers. Normally, HPV exists as a circular episomal DNA in the infected cell. However, in some instances, it integrates into the human genome in such a way as to enable increased expression of viral oncogenes, thereby leading to carcinogenesis. Since viral integration requires breaks in both viral and human genomes, DNA damage likely plays a key role in this critical process. One potentially significant source of DNA damage is exposure to elevated doses of ionizing radiation. Natural background radiation is ubiquitous; however, some populations, including radiological workers, radiotherapy patients, and astronauts, are exposed to significantly higher radiation doses, as well as to different types of radiation such as particle radiation. We hypothesize that ionizing radiation-induced DNA damage facilitates the integration of HPV into the human genome, increasing the risk of developing HPV-related cancers in the exposed population. To test this, we first determined the kinetics of DNA damage in keratinocytes exposed to ionizing radiation (protons) by assessing γ-H2AX foci formation using immunofluorescence (direct damage), and also measured ROS and 8-oxoG levels DCFDA and Avidin-FITC (indirect damage).As anticipated, direct DNA damage was observed promptly, within 30 min, whereas indirect DNA damage was delayed due to the time required for ROS to accumulate and cause oxidative damage. Although radiation was lethal at high doses, we were able to establish an experimental system where radiation exposure (protons and X-rays) induced DNA damage dose-dependently without causing major cytotoxic effects as assessed by several cytotoxicity assays. Most importantly, we explored the impact of radiation exposure on integration frequency using a clonogenic assay and demonstrated that as predicted, proton-induced DNA damage promotes the integration of HPV-like foreign DNA in oral keratinocytes. Overall, the insights gained from this work enable us to better understand the contribution of radiation exposure and DNA damage to HPV-mediated carcinogenesis and direct us toward strategies aimed at preventing malignancies in HPV-infected individuals.
高危型人乳头瘤病毒（HPV）几乎引发了所有宫颈癌病例，还与其他类型的肛门生殖器癌和口咽癌有关。通常，HPV在受感染细胞中以环状游离DNA的形式存在。然而，在某些情况下，它会以一种能使病毒癌基因表达增加的方式整合到人类基因组中，从而导致癌变。由于病毒整合需要病毒和人类基因组都发生断裂，DNA损伤可能在这一关键过程中起关键作用。DNA损伤的一个潜在重要来源是暴露于高剂量的电离辐射。天然本底辐射无处不在；然而，一些人群，包括放射工作人员、放疗患者和宇航员，会暴露于明显更高的辐射剂量以及不同类型的辐射，如粒子辐射。我们推测，电离辐射诱导的DNA损伤会促进HPV整合到人类基因组中，增加暴露人群患HPV相关癌症的风险。为了验证这一点，我们首先通过免疫荧光法评估γ-H2AX灶形成（直接损伤）来确定暴露于电离辐射（质子）的角质形成细胞中DNA损伤的动力学，还使用DCFDA和抗生物素蛋白-异硫氰酸荧光素测量了活性氧（ROS）水平和8-氧代鸟嘌呤（8-oxoG）水平（间接损伤）。正如预期的那样，直接DNA损伤在30分钟内迅速出现，而间接DNA损伤由于ROS积累和导致氧化损伤所需的时间而延迟。虽然高剂量辐射是致命的，但我们能够建立一个实验系统，在该系统中，通过几种细胞毒性试验评估，辐射暴露（质子和X射线）能剂量依赖性地诱导DNA损伤，而不会引起主要的细胞毒性作用。最重要的是，我们使用克隆形成试验探究了辐射暴露对整合频率的影响，并证明正如所预测的那样，质子诱导的DNA损伤促进了HPV样外源DNA在口腔角质形成细胞中的整合。总体而言，这项工作获得的见解使我们能够更好地理解辐射暴露和DNA损伤对HPV介导的癌变的作用，并指导我们制定旨在预防HPV感染个体发生恶性肿瘤的策略。
