Roger F. Martin

Use of the γ-H2AX assay to monitor DNA damage and repair in translational cancer research

Formation of γ-H2AX in response to DNA double stranded breaks (DSBs) provides the basis for a sensitive assay of DNA damage in human biopsies. The review focuses on the application of γ-H2AX-based methods to translational studies to monitor the clinical response to DNA targeted therapies such as some forms of chemotherapy, external beam radiotherapy, radionuclide therapy or combinations thereof. The escalating attention on radiation biodosimetry has also highlighted the potential of the assay including renewed efforts to assess the radiosensitivity of prospective radiotherapy patients. Finally the γ-H2AX response has been suggested as a basis for an in vivo imaging modality.

γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis.

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.

Induction of Double-strand Breaks Following Neutron Capture by DNA-bound 157Gd

Irradiation of plasmid DNA/Gd3+ mixtures with thermal neutrons induces DNA double-strand breaks (dsb). However, the extent of breakage is markedly reduced by sequestering the Gd3+ from DNA by addition of EDTA. Since the 157Gd neutron capture event involves some internal conversion, we suggest that the DNA dsb induction results from Auger electron emission.

Modelling of Auger-Induced Dna Damage by Incorporated125I

We have analyzed a newly available high resolution and precision repeat of the original Martin and Haseltine experiment which includes the influence of DMSO on the results. The new model includes the production and diffusion of radical species and .OH radical attack on DNA as well as the direct hits. Calculations of single-strand breaks use individual Auger electron along with the tracks of electrons and radical species superimposed on an atomistic model of B-DNA. Comparison of the preliminary calculations with the experiment supports the earlier choice of data for the amount of energy required to produce a single-strand break, i.e. 17.5 eV. In a separate simulation we found that an average of less than two ionizations inducing a single-strand break gave the best fit to experimental data. Direct hits were found to be predominantly occurring at short range while the damage by .OH radicals was mainly of the long-range type.

Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. II. The Role of Auger Electron Irradiation Compared to Charge Neutralization in DNA Breakage

Abstract Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. II. The Role of Auger Electron Irradiation Compared to Charge Neutralization in DNA Breakage. The dramatic chemical and biological effects of the decay of DNA-incorporated 125I stem from two consequences of the Auger electron cascades associated with the decay of the isotope: high local deposition of radiation energy from short-range Auger electrons, and neutralization of the multiply charged tellurium atom. We have analyzed the extensive data reported in the companion paper (Radiat. Res. 153, 000–000, 2000), in which DNA breakage was measured after 125I decay in a 41-bp oligoDNA. The experimental data collected under scavenging conditions (2 M dimethylsulfoxide) were deconvoluted into two components denoted as radiation and nonradiation, the former being attributed to energy deposition by Auger electrons. The contribution of the components was estimated by adopting various assumptions, the principal one being that DNA breakage due to the radiation mechanism is dependent on the distance between the decaying 125I atom and the cleaved deoxyribosyl unit, while the nonradiation mechanism, associated with neutralization of the multiply charged tellurium atom, contributes equally at corresponding nucleotides starting from the 125I-incorporating nucleotide. Comparison of the experimental data sets collected under scavenging and nonscavenging (without dimethylsulfoxide) conditions was used to estimate the radiation-scavengeable component. Our analysis showed that the nonradiation component plays the major role in causing breakage within 4–5 nucleotides from the site of 125I incorporation and produces about 50% of all single-stranded breaks. This overall result is consistent with the relative amounts of energy associated with Auger electrons and the charged tellurium atom. However, the nonradiation component accounts for almost four times more breaks in the top strand, to which the 125I is bound covalently, than in the bottom strand, thus suggesting an important role of covalent bonds in the energy transfer from the charged tellurium atom. The radiation component dominates at the distances beyond 8–9 nucleotides, and 36% of the radiation-induced breaks are scavengeable.

Sequence specificity of 125I-labelled Hoechst 33258 in intact human cells☆

Using polyacrylamide/urea DNA sequencing gels, the DNA sequence selectivity of 125I-labelled Hoechst 33258 damage has been determined in intact human cells to the exact base-pair. This was accomplished using a novel procedure with human alpha RI-DNA as the target DNA sequence. In this procedure, after size fractionation, the alpha RI-DNA is selectively purified by hybridization to a single-stranded M13 clone containing an alpha RI-DNA insert. The sequence specificity of [125I]Hoechst 33258 was indistinguishable in intact cells from purified high molecular weight DNA; and this is surprising considering the more complex environment of DNA in the nucleus where DNA is bound to nucleosomes and other DNA binding proteins. The ligand preferentially binds to DNA sequences which have four or more consecutive A.T base-pairs. The extent of damage was measured with a densitometer and, relative to the damage hotspot at base-pair 94, the extent of damage was similar in both purified high molecular weight DNA and intact cells. [125I]Hoechst 33258 causes only double-strand breaks, since single-strand breaks or base damage were not detected. These experiments represent the first occasion that the sequence specificity of a DNA damaging agent, which causes only double-strand breaks, has been determined to the exact base-pair in intact cells.

Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. I. Fragment Size Distribution and Evaluation of Breakage Probability

Abstract Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. I. Fragment Size Distribution and Evaluation of Breakage Probability. Incorporation of 125I-dC into a defined location of a double-stranded oligodeoxynucleotide was used to investigate DNA breaks arising from decay of the Auger electron-emitting isotope. Samples of the oligodeoxynucleotide were also labeled with 32P at either the 5′ or 3′ end of either the 125I-dC-containing (so-called top) or opposite (bottom) strand and incubated in 20 mM phosphate buffer or the same buffer plus 2 M dimethylsulfoxide at 4°C during 18–20 days. The 32P-end-labeled fragments produced by 125I decays were separated on denaturing polyacrylamide gels, and the 32P activity in each fragment was determined by scintillation counting after elution of fragments from the gel. The relative fragment size distributions were then normalized on a per decay basis and converted to a distribution of single-strand break probabilities as a function of distance from the 125I-dC. The results of three to five experiments for each of eight possible combinations of labels and incubation conditions are presented as a table showing the relative numbers of 32P counts in different fragments as well as graphs of normalized fragment size distributions and probabilities of breakage. The average numbers of single-strand breaks per 125I decay are 3.3 and 3.7 in the top strand and 1.3 and 1.5 in the bottom strand with and without dimethylsulfoxide, respectively. Every 125I decay event produces a break in the top strand, and breakage of the bottom strand occurs in 75–80% of the events. Thus a double-strand break is produced by 125I decay with a probability of approximately 0.8.

In Vitro Studies with Methylproamine A Potent New Radioprotector

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is ∼100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)2 confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.

Pulse radiolysis studies indicate that electron transfer is involved in radioprotection by hoechst 33342 and methylproamine

PURPOSE The aim of the study was to obtain evidence to support the hypothesis that the radioprotection by DNA-binding bibenzimidazoles is due to reduction by the DNA-bound ligand of transient radiation-induced oxidizing species on DNA, by following oxidation of the ligand after pulse radiolysis. A second aim was to compare the activities of methylproamine and Hoechst 33342 in the pulse radiolysis system, with the view to seeking a correlation with radioprotective activity. METHODS Solutions of deoxyguanosine or DNA, with or without Hoechst 33342 or methylproamine, and containing sodium selenate and tert-butanol were subjected to pulse radiolysis, and the oxidation of the ligand followed by time-resolved spectrophotometry. RESULTS The initial pulse radiolysis experiments using deoxyguanosine (dG) established that pulse radiolysis of sodium selenate produces a transient oxidant SeO3*-, which oxidizes dG to a species (presumably dG*+), with spectral characteristics indistinguishable from those described in previous pulse radiolysis studies using Br2*- as the oxidant. The estimate obtained for the bimolecular rate constant (k2) for the reaction of the selenite radical with dG, was 1.2 x 10(9) M(-1) s(-1). The corresponding reaction of SeO3*- with DNA is much slower (k2 3 x 10(7) M(-1) s(-1)). Although unbound Hoechst 33342 is oxidized directly by SeO3*- (k2 2.3 x 10(9) M(-1) s(-1)), experiments with mixtures of Hoechst 33342 with an excess of dG (or DNA) indicated that ligand oxidation was mediated by dG*+ (or DNAoxid). For example, successive dilution of a DNA-Hoechst solution had little impact on the rate of ligand oxidation, consistent with an intramolecular rate-determining step. When the concentration of DNA was maintained at 1.0 mM DNA bp, increasing the concentration of the ligand resulted in a linear increase in the rate of oxidation; the increase being steeper for methylproamine than for Hoechst 33342. Investigation of the dependence of yield of oxidized ligand on ligand occupancy also indicated that the methylproamine was more active than Hoechst 33342, with the estimates for the range of electron transfer from the ligand to DNAoxid being 14 and 31 bp for Hoechst 33342 and methylproamine, respectively. CONCLUSIONS At this stage we conclude that radioprotection by these DNA-binding ligands is mediated by electron transfer, and that the improved radioprotective activity of methylproamine may be attributable to the observed kinetic differences. However, further studies are required to confirm the correlation, and if it is sustained, pulse radiolysis could be useful in evaluating new analogues in an attempt to further improve the radioprotective properties of methylproamine, which already has considerable clinical potential.

Plasmid DNA Breakage by Decay of DNA-Associated Auger Electron Emitters: Approaches to Analysis of Experimental Data

Abstract Lobachevsky, P. N., Karagiannis, T. C. and Martin, R. F. Plasmid DNA Breakage by Decay of DNA-Associated Auger Electron Emitters: Approaches to Analysis of Experimental Data. Radiat. Res. 162, 84–95 (2004). Plasmid DNA is a popular substrate for the assay of DNA strand breakage by a variety of agents. The use of the plasmid assay relies on the assumption that individual damaging events occur at random, which allows the application of Poisson statistics. This assumption is not valid in the case of damage arising from decay of DNA-associated Auger electron emitters, since a single decay event can generate a few breaks in the same DNA strand, which is indistinguishable from a single break in the assay. The consequent analytical difficulties are overcome by considering relaxation events rather than single-strand breaks, and linearization events rather than double-strand breaks. A further consideration is that apart from damage at the site of DNA-associated decay, which is the principal interest of the analysis, some DNA damage also arises from the radiation field created by all decay events. These two components of damage are referred to as internal and external breakage, respectively, and they can be separated in the analysis since their contribution depends on the experimental conditions. The DNA-binding ligand Hoechst 33258 labeled with 125I was used in our experiments to study breakage in pBR322 plasmid DNA arising from the decay of this Auger electron emitter. The values obtained for the efficiency (per decay) of plasmid relaxation and linearization by the 125I-labeled ligand were 0.090 ± 0.035 and 0.82 ± 0.04, respectively. When dimethylsulfoxide was included as a radical scavenger, the efficiency values for relaxation and linearization were 0.15 ± 0.02 and 0.65 ± 0.05, respectively.