γH2AX is a biomarker of modulated cytostatic drug resistance.

Abstract

ONE reason for reduced therapeutic efficiency of cancer chemotherapies is the appearance of multidrug resistance. Plasma membrane glycoprotein (P-gp) is recognized as one of the best characterized drug efflux pumps playing a central role in the absorption and disposition of many drugs, including chemotherapeutic agents. Increased P-gp activity has been suggested and reported to be an indicator of chemotherapeutic failure. Therefore, P-gp activity has been used as a potential target in being able to reverse multidrug resistance. Cytostatic drugs such as etoposide in combination with the immunosuppressive cyclosporine A may inhibit P-gp activity, ultimately resulting in intracellular enrichment of the targeted drug (1). One issue in cancer therapy involves the highly varying drug response among patients. To assess the multidrug resistance status for personalized cancer therapy, an assay that can provide results to monitor the cytostatic effect of antineoplastic agents in combination with different drugs is highly desirable. Many of these drugs, like etoposide, cause DNA double strand breaks (DSBs) that may eventuate in clonogenic death. DSBs are one of the most biologically significant DNA damage lesions that leads to chromosome breakage and/or rearrangement, mutagenesis and loss or gain of genetic information (2). The phosphorylation of H2AX histone proteins which are located in the vicinity of the DSBs is known as one of the earliest responses to DNA DSBs in cells. Induction of DNA DSBs in live mammalian cells triggers the phosphorylation of Ser139 in the SQ motif near the C-terminal of H2AX protein, which results in the phosphorylated form of H2AX, termed cH2AX. cH2AX signals appear as discrete nuclear “foci” which can be visualized and quantified by a number of methods including fluorescence microscopy and flow cytometry, following immunostaining procedures with fluorescence-coupled antibodies. The cH2AX foci counting approach has been used in numerous studies to assess the relationship between cH2AX foci removal and the rate of DSBs repair (3). Each DSB is represented by an individual cH2AX focus with a ratio of 1:1 and after successful repair of DSBs, the level of cH2AX foci decline over time in the presence of normal physiological repair mechanisms. The presence of persistent cH2AX indicates impaired DNA repair. Therefore, cH2AX quantification may prove to be a sensitive biomarker of DNA DSBs in human cells and has been suggested for biomonitoring tumor progression and treatment response (4). DSBs are directly generated by exogenous agents such as ionizing radiation and antitumor drugs (bleomycin, mitoxantrone, etoposide). Because mammalian cells respond to DSBs by activating a multitude of proteins involved in signalling and DNA repair pathways, the very nature of DSBs poses such a threat to cell survival that DNA damage checkpoint proteins may be activated to initiate cellular division arrest. This provides time for DNA repair to proceed before mitosis is completed or in the case of overwhelming damage, apoptosis ensues (5). Therefore, detection of accumulated cH2AX foci may be an efficient method for assessing multidrug resistance in cancer therapy as presented by Reddig et al. in this issue of Cytometry A (page XXX).