Meessen Sabine, Najjar Gregoire, Azoitei Anca, Iben Sebastian, Bolenz Christian, Günes Cagatay
Department of Urology, Ulm University Hospital, 89081 Ulm, Germany.
Department of Dermatology, Ulm University Hospital, 89081 Ulm, Germany.
Cancers (Basel). 2022 Apr 28;14(9):2205. doi: 10.3390/cancers14092205.
Aberrant replication stress (RS) is a source of genome instability and has serious implications for cell survival and tumourigenesis. Therefore, the detection of RS and the identification of the underlying molecular mechanisms are crucial for the understanding of tumourigenesis. Currently, three protein markers-p33-phosphorylated replication protein A2 (pRPA2), γ-phosphorylated H2AX (γ-H2AX), and Tumor Protein P53 Binding Protein 1 (53BP1)-are frequently used to detect RS. However, to our knowledge, there is no report that compares their suitability for the detection of different sources of RS. Therefore, in this study, we evaluate the suitability of pRPA2, γ-H2AX, and 53BP1 for the detection of RS caused by different sources of RS. In addition, we examine their suitability as markers of the telomerase-mediated alleviation of RS. For these purposes, we use here telomerase-negative human fibroblasts (BJ) and their telomerase-immortalized counterparts (BJ-hTERT). Replication stress was induced by the ectopic expression of the oncogenic RAS mutant RAS (OI-RS), by the knockdown of ploidy-control genes ORP3 or MAD2 (AI-RS), and by treatment with hydrogen peroxide (ROS-induced RS). The level of RS was determined by immunofluorescence staining for pRPA2, γ-H2AX, and 53BP1. Evaluation of the staining results revealed that pRPA2- and γ-H2AX provide a significant and reliable assessment of OI-RS and AI-RS compared to 53BP1. On the other hand, 53BP1 and pRPA2 proved to be superior to γ-H2AX for the evaluation of ROS-induced RS. Moreover, the data showed that among the tested markers, pRPA2 is best suited to evaluate the telomerase-mediated suppression of all three types of RS. In summary, the data indicate that the choice of marker is important for the evaluation of RS activated through different conditions.
异常复制应激（RS）是基因组不稳定的一个来源，对细胞存活和肿瘤发生具有严重影响。因此，检测RS并确定其潜在分子机制对于理解肿瘤发生至关重要。目前，三种蛋白质标志物——p33-磷酸化复制蛋白A2（pRPA2）、γ-磷酸化H2AX（γ-H2AX）和肿瘤蛋白P53结合蛋白1（53BP1）——经常用于检测RS。然而，据我们所知，尚无报告比较它们对不同来源RS的检测适用性。因此，在本研究中，我们评估了pRPA2、γ-H2AX和53BP1对不同来源RS所致RS的检测适用性。此外，我们还研究了它们作为端粒酶介导的RS缓解标志物的适用性。为了这些目的，我们在此使用端粒酶阴性的人成纤维细胞（BJ）及其端粒酶永生化对应物（BJ-hTERT）。通过致癌RAS突变体RAS的异位表达（OI-RS）、通过敲低倍性控制基因ORP3或MAD2（AI-RS）以及用过氧化氢处理（ROS诱导的RS）来诱导复制应激。通过对pRPA2、γ-H2AX和53BP1进行免疫荧光染色来确定RS水平。对染色结果的评估显示，与53BP1相比，pRPA2和γ-H2AX对OI-RS和AI-RS提供了显著且可靠的评估。另一方面，在评估ROS诱导的RS时，53BP1和pRPA2被证明优于γ-H2AX。此外，数据表明，在所测试的标志物中，pRPA2最适合评估端粒酶介导的对所有三种类型RS的抑制。总之，数据表明标志物的选择对于评估通过不同条件激活的RS很重要。
