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Review
. 2010 Sep 15;16(18):4532-42.
doi: 10.1158/1078-0432.CCR-10-0523. Epub 2010 Sep 7.

Histone gammaH2AX and poly(ADP-ribose) as clinical pharmacodynamic biomarkers

Christophe E Redon  1 , Asako J Nakamura, Yong-Wei Zhang, Jiuping Jay Ji, William M Bonner, Robert J Kinders, Ralph E Parchment, James H Doroshow, Yves Pommier
Affiliations
Review

Histone gammaH2AX and poly(ADP-ribose) as clinical pharmacodynamic biomarkers

Christophe E Redon et al. Clin Cancer Res. .

Abstract

Tumor cells are often deficient in DNA damage response (DDR) pathways, and anticancer therapies are commonly based on genotoxic treatments using radiation and/or drugs that damage DNA directly or interfere with DNA metabolism, leading to the formation of DNA double-strand breaks (DSB), and ultimately to cell death. Because DSBs induce the phosphorylation of histone H2AX (γH2AX) in the chromatin flanking the break site, an antibody directed against γH2AX can be employed to measure DNA damage levels before and after patient treatment. Poly(ADP-ribose) polymerases (PARP1 and PARP2) are also activated by DNA damage, and PARP inhibitors show promising activity in cancers with defective homologous recombination (HR) pathways for DSB repair. Ongoing clinical trials are testing combinations of PARP inhibitors with DNA damaging agents. Poly(ADP-ribosylation), abbreviated as PAR, can be measured in clinical samples and used to determine the efficiency of PARP inhibitors. This review summarizes the roles of γH2AX and PAR in the DDR, and their use as biomarkers to monitor drug response and guide clinical trials, especially phase 0 clinical trials. We also discuss the choices of relevant samples for γH2AX and PAR analyses.

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Figures

Figure 1
Figure 1. γH2AX following DSB formation
H2AX is phosphorylated by members of the phosphatidylinositol-3 kinase (PI3K) family; which one is involved depends on the type of genotoxic stress (4). While ATM and ATR are primarily involved in H2AX phosphorylation following IR and replication stress respectively, DNA-PK and JNK-1 were shown to be responsible for γH2AX formation during apoptosis (97, 98). γH2AX foci are known to be involved in the recruitment and stabilization of DDR proteins including Mre11, Rad50, Nbs1 (the MRN complex), MDC1, 53BP1, BRCA1, ATM, and RNF8 (6, 99-101). DSB repair is performed by the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. HR, driven by the BRCA2, RAD51 and RAD52/54 genes, is the more accurate because it utilizes a homologous DNA segment to act as a template for the damaged DNA region. Repair is also performed by sister chromatid-dependent recombination repair via cohesin recruitment (102, 103). In contrast, NHEJ is faster, does not require a homologous DNA segment, and can operate in non-replicating cells. However, it is error-prone. The classical effectors of NHEJ are the end-binding proteins Ku70/80, DNA-dependent protein kinase (DNA-PKcs), the nuclease Artemis, the scaffolding protein XRCC4 and ligase IV. Recently, a slow DNA-PK-independent NHEJ pathway involving PARP1, histone H1, XRCC1 and ligase III has been proposed (24, 25). The γH2AX foci are also involved in chromatin alteration via recruitment of remodeling complexes and in signal transduction (accrued ATM activation, G2/M cell cycle checkpoint). In addition, γH2AX foci, through their recruitment of the cohesions and the MRN complexes, are involved in binding and tethering the broken DNA ends may help prevent the dissociation of the broken chromosome ends (104).
Figure 2
Figure 2. Schematic representation of PARP1's role in single-strand break repair
Upon detection of the DNA single-strand break (SSB) lesion, PARP1 is activated and, in turn, synthesizes PAR polymers attached to itself and other acceptor proteins at the DNA lesion site (Histone H1, other core histones, TOP1,...). These accrued post-translational modifications favor the recruitment of other factors involved in DNA repair, especially those involved in the base excision repair (XRCC1, Tdp1, Ligase III, Polβ). If left unrepaired, for example with PARP inhibition (red arrows), SSBs can lead to DSBs and γH2AX foci formation. DSB repair requires BRCA1/2, proteins that are deficient in many, including breast and ovarian, cancers.
Figure 3
Figure 3. Human biopsy analysis
(A) Biopsy sites and methods used to analyze γH2AX levels and PARP activities. (B) Biosample accessibility, state of DNA metabolism, and expected γH2AX response. Note that the most accessible tissues for analysis are not always the most appropriate for measuring a drug response. For example, lymphocyte and oral cells, two highly differentiated cell types, may exhibit poor responses to chemotherapeutic drugs targeting DNA replication. In contrast, tumor cells, while clearly appropriate, are often poorly accessible particularly for repetitive sampling. γH2AX formation is independent of the cell cycle state, occurring in cancer cells as well as in lymphocytes and oral cells after irradiation (37, 40, 70). CTCs: circulating tumor cells.
Figure 4
Figure 4. γH2AX and PAR detection
γH2AX detection in lymphocytes (A), tumor needle biopsies (B), and plucked hairs (C) from patients undergoing chemotherapy. The white box in the lower panel (C) marks the region of active γH2AX formation in a typical plucked hair. Green, γH2AX; red, DNA. (D) Pharmacodynamic assay developed at the National Cancer Institute to measure PAR as a biomarker for PARP inhibition in both tumor biopsies and peripheral blood mononuclear cells.
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