Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovakia; Department of Medical Biophysics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Malá Hora 4, Martin, Slovakia.
Department of Radiobiology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovakia.
Toxicol In Vitro. 2021 Jun;73:105127. doi: 10.1016/j.tiv.2021.105127. Epub 2021 Feb 27.
While hyperthermia (HT) is a promising modality for cancer treatment, the knowledge on mechanisms of its effect on cells is still limited. We have investigated DNA double-strand break (DSB) and apoptosis induced by HT. Umbilical cord blood lymphocytes (UCBL) were subjected to HT at 43 °C. We have treated cells for 1 h (1 h HT), 2 h (2 h HT) and by combined HT and ice treatment (both lasting 1 h). Enumeration of DSB by 53BP1/γH2AX DNA repair focus formation and early apoptosis by γH2AX pan-staining was conducted by automated fluorescent microscopy. Apoptotic stages and viability were assessed by the annexin/propidium iodide (PI) assay using flow cytometry 0, 18, and 42 h post-treatment. HT induced either immediate (2 h HT) or postponed (1 h HT) DNA damage. The levels of 53BP1 and γH2AX foci differed under the same treatment conditions, suggesting that the ratio of co-localized γH2AX/53BP1 foci to all γH2AX and also to all 53BP1 foci could be a valuable marker. The ratio of co-localized foci increased immediately after 2 h HT regardless the way of assessment. For the first time we show, by both annexin/PI and γH2AX pan-staining assay that apoptosis can be induced during or immediately after the 2 h HT treatment. Our results suggest that HT may induce DSB in dependence on treatment duration and post-treatment time due to inhibition of DNA repair pathways and that HT-induced apoptosis might be dependent or associated with DSB formation in human lymphocytes. Assessment of γH2AX pan-staining in lymphocytes affected by HT may represent a valuable marker of HT treatment side effects.
虽然高热（HT）是一种有前途的癌症治疗方式，但关于其对细胞影响的机制的知识仍然有限。我们研究了 HT 诱导的 DNA 双链断裂（DSB）和细胞凋亡。将脐带血淋巴细胞（UCBL）在 43°C 下进行 HT。我们用 1 小时（1 h HT）、2 小时（2 h HT）和 HT 与冰处理联合（均持续 1 小时）处理细胞。通过自动化荧光显微镜对 53BP1/γH2AX DNA 修复焦点形成和通过γH2AX 全染色进行的早期凋亡进行 DSB 计数。用流式细胞术在 0、18 和 42 小时后用 Annexin/PI（PI）检测评估凋亡阶段和活力。HT 诱导即刻（2 h HT）或延迟（1 h HT）DNA 损伤。在相同的处理条件下，53BP1 和 γH2AX 焦点的水平不同，这表明共定位的 γH2AX/53BP1 焦点与所有 γH2AX 和所有 53BP1 焦点的比值可能是一个有价值的标记。无论评估方式如何，2 h HT 后即刻共定位焦点的比值均增加。我们首次通过 Annexin/PI 和 γH2AX 全染色试验表明，凋亡可在 2 h HT 治疗期间或之后立即诱导。我们的结果表明，HT 可能通过抑制 DNA 修复途径而在治疗持续时间和治疗后时间上诱导 DSB，并且 HT 诱导的凋亡可能与人类淋巴细胞中的 DSB 形成有关或与之相关。评估受 HT 影响的淋巴细胞中的γH2AX 全染色可能代表 HT 治疗副作用的一个有价值的标志物。
