title: Assessment of modulated cytostatic drug resistance by automated γH2AX analysis.
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..
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Reddig
authors.affiliation: Medipan GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lorenz
authors.affiliation: Faculty of Natural Sciences, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Rico
authors.initials: R
authors.lastname: Hiemann
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Karina
authors.initials: K
authors.lastname: Guttek
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Hartig
authors.affiliation: Medipan GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Heiserich
authors.affiliation: Medipan GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Eberle
authors.affiliation: Medipan GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Peters
authors.affiliation: Faculty of Natural Sciences, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schierack
authors.affiliation: Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Sack
authors.affiliation: Medipan GmbH, Dahlewitz/Berlin, Germany.|Faculty of Natural Sciences, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Roggenbuck
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Reinhold
mesh_terms: D000328:Adult; D016572:Cyclosporine / Q000494:pharmacology; D054697:Cytostatic Agents / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000235:genetics; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D018432:Drug Resistance, Multiple / Q000187:drug effects* / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology; D005260:Female; D006657:Histones / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D020123:Sirolimus / Q000494:pharmacology; D055815:Young Adult
chemical_list: D054697:Cytostatic Agents; C468783:H2AX protein, human; D006657:Histones; D005047:Etoposide; D016572:Cyclosporine; D004247:DNA; D020123:Sirolimus
keywords: DNA double-strand breaks; MDR modulation; P-gp inhibition; automated γH2AX foci analysis; cytostatic drug resistance; drug efflux; etoposide