Reddig Annika, Lorenz Sebastian, Hiemann Rico, Guttek Karina, Hartig Roland, Heiserich Lisa, Eberle Caroline, Peters Vanessa, Schierack Peter, Sack Ulrich, Roggenbuck Dirk, Reinhold Dirk
Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
Medipan GmbH, Dahlewitz/Berlin, Germany.
Cytometry A. 2015 Aug;87(8):724-32. doi: 10.1002/cyto.a.22667. Epub 2015 Apr 2.
The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double-strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug-induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter-individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P-gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB-inducing agent etoposide (ETP) are known P-gp substrates. In this respect, modulation of MDR by P-gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB-positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB-inducing cytostatics..
许多化疗药物的疗效依赖于通过诱导各种DNA损伤来优先破坏快速分裂的癌细胞。最有害的DNA损伤类型是DNA双链断裂（DSB），可通过磷酸化组蛋白H2AX（γH2AX）的免疫荧光染色来检测。此外，γH2AX已被认为是化疗癌症治疗中的临床药效学生物标志物。治疗肿瘤疾病的一个巨大挑战是癌症患者之间不同的反应行为。因此，外排泵的内在或药物诱导的过表达通常会导致多药耐药（MDR）和治疗失败。特别是，外排泵（如通透糖蛋白（P-gp））表达水平的个体差异与癌症进展相关。几种有效的细胞毒性药物，包括诱导DSB的药物依托泊苷（ETP），都是已知的P-gp底物。在这方面，已经描述了P-gp抑制剂（如免疫抑制剂环孢素A（CsA）和雷帕霉素（Rapa））对MDR的调节作用。在此，我们研究了γH2AX焦点分析在监测CsA和Rapa对人外周血单个核细胞中ETP诱导的细胞毒性影响方面的应用。通过自动荧光显微镜和解释系统AKLIDES对γH2AX焦点进行评估。与单独使用ETP治疗相比，我们的结果显示，在存在CsA或Rapa的情况下，细胞用ETP处理后，γH2AX焦点数量和DSB阳性细胞百分比显著增加。相比之下，单独用CsA或Rapa孵育的细胞的DSB水平与未处理细胞的焦点数量相当。我们的结果成功证明了自动γH2AX分析如何可以作为一种快速可靠的方法来监测耐药性以及在使用诱导DSB的细胞毒性药物治疗期间MDR调节剂的影响。
