Niechoda Agata, Milewska Katarzyna, Roslan Joanna, Ejsmont Karolina, Holownia Adam
Department of Pharmacology, Medical University of Bialystok, Bialystok, Poland.
Front Physiol. 2023 Aug 14;14:1238150. doi: 10.3389/fphys.2023.1238150. eCollection 2023.
Exposure to particulate matter is associated with DNA damage and the risk of lung cancer. Protein p53 is activated by multi-site phosphorylation in the early stages of DNA damage and affects cell outcome. Our study aimed to assess the effect of (100 µg/mL/24 h) standardized air pollutants: carbon black (CB), urban dust (UD), and nanoparticle carbon black (NPCB) on cell cycle, DNA damage and 53 phosphorylation at Ser 9, Ser 20, Ser 46, and Ser 392 in proliferating and quiescent A549 cells and in cells that survived cisplatin (CisPT) exposure. Phosphorylated p53 was quantified in cell subpopulations by flow cytometry using specific fluorochrome-tagged monoclonal antibodies and analysis of bivariate fluorescence distribution scatterplots. CisPT, UD and NPCB increased site-specific p53 phosphorylation producing unique patterns. NPCB activated all sites irrespectively on the cell cycle, while the UD was more selective. p53 Ser 9-P and p53 Ser 20-P positively correlated with the numbers of CisPT-treated cells at G0/G1, and NPCB and NPCB + CisPT produced a similar effect. A positive correlation and integrated response were also found between Ser 20-P and Ser 392-P in resting A549 cells treated with NPCB and CisPT but not UD. Interdependence between the expression of p53 phosphorylated at Ser 20, and Ser 392 and cell cycle arrest show that posttranslational alterations are related to functional activation. Our data suggest that p53 protein phosphorylation in response to specific DNA damage is driven by multiple independent and integrated pathways to produce functional activation critical in cancer prevention and treatment.
接触颗粒物与DNA损伤及肺癌风险相关。蛋白质p53在DNA损伤早期通过多位点磷酸化被激活，并影响细胞结果。我们的研究旨在评估（100μg/mL/24h）标准化空气污染物：炭黑（CB）、城市灰尘（UD）和纳米颗粒炭黑（NPCB）对增殖和静止的A549细胞以及顺铂（CisPT）暴露后存活细胞的细胞周期、DNA损伤和p53在丝氨酸9、丝氨酸20、丝氨酸46和丝氨酸392位点磷酸化的影响。使用特异性荧光染料标记的单克隆抗体通过流式细胞术对细胞亚群中的磷酸化p53进行定量，并分析双变量荧光分布散点图。CisPT、UD和NPCB增加了位点特异性p53磷酸化，产生独特模式。NPCB在细胞周期的各个阶段均激活所有位点，而UD则更具选择性。p53丝氨酸9磷酸化（p53 Ser 9-P）和p53丝氨酸20磷酸化（p53 Ser 20-P）与处于G0/G1期的CisPT处理细胞数量呈正相关，NPCB和NPCB + CisPT产生类似效果。在用NPCB和CisPT而非UD处理的静止A549细胞中，丝氨酸20磷酸化（Ser 20-P）和丝氨酸392磷酸化（Ser 392-P）之间也发现了正相关和综合反应。丝氨酸20磷酸化、丝氨酸392磷酸化的p53表达与细胞周期停滞之间的相互依赖性表明，翻译后改变与功能激活相关。我们的数据表明，响应特定DNA损伤的p53蛋白磷酸化由多个独立且相互整合的途径驱动，以产生对癌症预防和治疗至关重要的功能激活。
