Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
γ-H2AX and other histone post-translational modifications in the clinic☆
Highlights
► We review the uses of γ-H2AX and other modified histone species in the clinic. ► γ-H2AX is a useful radiation biodosimeter. ► γ-H2AX can be used to assay DNA damage during chemotherapy. ► γ-H2AX can be used to optimize cancer treatments and other procedures. ► Uses of other modified histone species are discussed.
Introduction
Much of the research on the origins of cancer has focused on cellular DNA since alterations in its sequence, of one kind or another, appear to be central to tumorigenesis. In some cases, these changes are manifested in genomic alterations involving chromosome rearrangements, duplications, and deletions, and may have originated from misrepaired DNA double-strand breaks (DSBs). These serious ramifications make the DSB among the most serious of DNA lesions.
DNA lesions occur in the context of chromatin, an organized structure of DNA and proteins consisting of fundamental units called nucleosomes, which contain DNA coils of 145–147 bp. The nucleosomes are spaced along the continuous DNA double-helix with varying lengths of DNA linking them [1]. The nucleosome consists of an octamer of histone proteins that form a core for the DNA coil. The linker is also a complex of DNA and proteins including other histones which spans the gap between adjacent nucleosomes while maintaining a continuous DNA double helix [1]. Compared to naked DNA, the DNA in the nucleosome is compacted ~ 6:1. Further organization of nucleosomes into 30 nm filaments increases the packing ratio to ~ 40:1. Looping of the filaments brings the ratio to ~ 1000:1 in interphase chromatin, and mitotic condensation brings the ratio to ~ 10,000:1. Thus the repair of DNA lesions also occurs in the context of a highly dynamic complex structure.
The histone structures are highly conserved, consistent with the notion that these proteins play similar roles throughout evolution. Nucleosomal histones belong to four families, H2A, H2B, H3 and H4, while the linker histones are from one family, H1 [2], [3]. Each histone family contains multiple genes [4], most of which encode identical or almost identical proteins [5]. These differences are thought to reflect selective pressure rather than differential function [5]. However, in some families, several genes encode proteins with unique functions. Notably, the H2A family contains two species, H2AZ and H2AX, which are present throughout eukaryotic evolution from yeast to human [5]. Each nucleosomal octamer contains two molecules of each of the four families noted earlier. However, while the factors determining the relative abundance of the different gene products in each family are obscure, H2AZ and H2AX are found in a substantial minority of the H2A positions in mammals [5], [6].
Section snippets
Histone post-translational modifications (PTMs)
Histone post-translational modifications (PTMs) are dynamic biological sensors that adjust in response to endogenous and/or exogenous cellular stress [7], [8], [9], [10]. Because some of these stresses can be associated with diseases such as cancer, identifying alterations in histone PTM homeostatic levels may yield valuable clinical information on treatment efficacy and/or the condition of a disease [10], [11] (Fig. 1). An increasing body of studies suggests that these histone PTMs, also
H2AX and DNA DSBs
While erroneous repair of DSBs is a major source of genomic instability and can lead ultimately to cancer, many cancer therapies induce DSBs as a way to kill cancer cells. For this reason, specific and sensitive in vivo biomarkers for DSB formation enable the responses of individual patients to these therapies to be monitored. The unique function of H2AX lies with the phosphorylation of a serine four residues from the C-terminus (C-4) [20], in a consensus sequence which has been conserved
γ-H2AX as a biomarker for DNA DSBs in vivo
Cancer treatment may be improved if more information was available on the responses of individual patients to particular protocols. Currently, patient responses to treatments may be unknown for several weeks until tumor size is assessed by various imaging techniques [34]. By one criterion, RECIST, a 30% decrease in tumor diameters is considered a partial response. The Choi criterion also takes account of changes in tumor density. A more recent criterion for evaluating patient responses is
γ-H2AX assay types
Detection of γ-H2AX, utilizing a variety of assays, is being used beyond basic research as a biomarker for cancer, a biodosimeter for radiation exposure and drug development, and a tool to identify genotoxic compounds in occupational or environmental studies [36] (Fig. 4). However, microscopy is still the prevailing method for γ-H2AX detection for clinical applications since it is the most sensitive of the possible approaches, capable of detecting a single DSB [23]. Solid tissues can be
Biological samples for γ-H2AX assays
Because cell proliferation is different among human tissues (i.e. higher proliferation rates in intestine, and bone marrow), the choice of biospecimens is critical for studies of drugs that target DNA metabolism. In contrast, protocols using radiation (i.e. radiotherapy) may produce DSBs more homogeneously throughout the human body, independently of DNA metabolism. There are several possible choices for patient tissue samples, each with its advantages and issues [38]. One option is to obtain
Applications: γ-H2AX as a radiation biodosimeter
The most obvious application of γ-H2AX measurements is the assessment of DNA damage from ionizing radiation. The radiation can be external as during typical radiation treatments or internal as during radioisotope therapy, where radioisotopes are given by infusion or ingestion. A non-exhaustive list of clinical studies utilizing γ-H2AX as a biodosimeter is presented in Table 1.
Applications: the use of γ-H2AX in drug efficacy measurements
There are many therapeutic strategies to combat cancer. These include among others, hormonal therapy to impede growth signaling through hormone receptors on cancer cells, immunotherapy to boost the immune system to increase cancer cell killing, targeted therapy to thwart growth signaling pathways in cancer cells using small enzyme inhibitors or antibodies, and chemotherapy to interfere with cell division and DNA metabolism [80]. As with radiotherapy, chemotherapy can act by generating
Applications: γ-H2AX and clinical diagnostics
In addition to its use in the clinic as a marker for both radiation biodosimetry and drug development for cancer research, recent studies have also demonstrated the putative use of γ-H2AX for medical diagnosis (Table 3). The following section will describe how the presence of γ-H2AX residual foci have been correlated with radiosensitivity in individuals as well as the potential of γ-H2AX to analyze cancer progression or the impact of other diseases on genome integrity.
Other histone post-translational modifications used as biomarkers in the clinic
Deregulation in any of the histone PTMs may alter gene expression and lead to changes in these cellular processes resulting in tumorigenesis [107], [108]. Similarly, alterations in the activities of histone-modifying enzymes such as histone deacetylases and histone methyltransferases have been linked to cancer [109]. A reasonable hypothesis is that exogenous and endogenous stresses may result in the alteration of specific types of histone PTMs, facilitating oncogenesis (Fig. 1). Pressure on
Concluding remarks
The development of agents for cancer treatment is a slow and laborious process. Despite many successes in the discovery of new anti-cancer drugs, only 5% of prospective drugs gain final approval [122]. The discovery and use of new biomarkers, such as γ-H2AX, would enable clinicians to evaluate a drug's effectiveness in a particular patient more quickly, reducing the time and cost of drug development, i.e., “fail fast”. The use of γ-H2AX has already aided studies of the effects of many drugs
Acknowledgements
This research was supported by the NIAID Radiation/Nuclear Countermeasures Program and the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, NIH.
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This article is part of a Special Issue entitled: Chromatin in time and space.