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Updated: Apr 15, 2026
An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes
Published on: December 25, 2021
Assessment of modulated cytostatic drug resistance by automated γH2AX analysis
Annika Reddig1, Sebastian Lorenz2, Rico Hiemann3
1Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
Abstract:
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..
Insights
This study shows that automated analysis of DNA double-strand breaks (DSB) using γH2AX foci can effectively monitor how drug resistance modulators impact chemotherapy effectiveness in cancer cells.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Chemotherapy efficacy depends on inducing DNA damage, particularly DNA double-strand breaks (DSB), in rapidly dividing cancer cells.
- Multiple Drug Resistance (MDR), often due to efflux pumps like P-glycoprotein (P-gp), is a major challenge in cancer treatment, leading to variable patient responses.
- P-gp substrates, such as etoposide (ETP), are cytostatic drugs, and their efficacy can be modulated by P-gp inhibitors like cyclosporine A (CsA) and rapamycin (Rapa).
Purpose of the Study:
- To investigate the utility of the γH2AX focus assay for monitoring the impact of P-gp inhibitors (CsA and Rapa) on etoposide (ETP)-induced cytotoxicity.
- To evaluate the effectiveness of automated fluorescence microscopy in assessing drug resistance and the effects of MDR modulators.
Main Methods:
- Human peripheral blood mononuclear cells were treated with ETP alone or in combination with CsA or Rapa.
- DNA double-strand breaks were detected and quantified by immunofluorescence staining of phosphorylated histone H2AX (γH2AX).
- Automated analysis of γH2AX foci was performed using the AKLIDES system.
Main Results:
- Treatment with ETP in the presence of CsA or Rapa significantly increased the number of γH2AX foci and the percentage of DSB-positive cells compared to ETP alone.
- Cells treated with CsA or Rapa alone showed DSB levels comparable to untreated cells, indicating no inherent cytotoxicity.
- Automated γH2AX analysis provided a rapid and reliable method for assessing drug resistance.
Conclusions:
- Automated γH2AX focus analysis is a valuable tool for monitoring the impact of MDR modulators on the efficacy of DSB-inducing chemotherapeutic agents.
- This approach can help personalize cancer treatment by evaluating individual responses to drug combinations and resistance mechanisms.
- The study highlights the potential of γH2AX as a pharmacodynamic biomarker in clinical settings for optimizing cancer therapy.
