[en] In summary, the major features of the data are: the breakage probability is close to 1 at the ¹²⁵IdC site, but declines rapidly at the neighbouring sites; the probability breakage somewhere in the bottom strand is 0.8, which determines the probability of a double-strand break; DSMO has negligible effect on breakage at sites within a few nucleotides of the ¹²⁵IdC. Although it has a substantial effect at more distant sites, the impact of DMSO on the overall yield of breakage is small. (authors)

[en] Full text: A double-stranded oligodeoxynucleotide containing ¹²⁵I-dC in a defined location, with 5'- or 3'-³²P-end-labelling of either strand, was used to investigate DNA strand breakage resulting from ¹²⁵I decay. Samples of the ³²P-end-labelled and ¹²⁵I-dC containing oligoDNA were incubated in 20 mM phosphate buffer (PB), or PB + 2 M dimethylsulphoxide (DMSO) at 4 deg during 18-20 days. The ³²P-end-labelled DNA fragments produced by ¹²⁵I decays were separated on denaturing polyacrylamide gels, and the ^3P activity in each fragment was determined by scintillation counting after elution from the gel. The fragment size distribution was then converted to a distribution of single stranded break probabilities at each nucleotide position. The results indicate that each ¹²⁵I decay event produces at least one break in the ¹²⁵I-dC containing strand, and causes breakage of the opposite strand in 75-80% of events. Thus, the double stranded break is produced by ¹²⁵I decay with probability ∼0.8. Most of single stranded breaks (around 90%) occurred within 5-6 nucleotides of the ¹²⁵I-dC, however DNA breaks were detected up to 18-20 nucleotides from the decay site. The average numbers of single stranded breaks per decay are 3.7 (PB) and 3.3 (PB+DMSO) in ¹²⁵I-dC containing strand, and 1.5 (PB) and 1.3 (PB+DMSO) in the opposite strand. Deconvolution of strand break probabilities as a function of separation from the ¹²⁵I, in terms of both distance (to target deoxyribosyl carbon atoms, in B-DNA) and nucleotide number, show that the latter is an important parameter for the shorter-range damage. This could indicate a role for attenuation/dissipation of damage through the stacked bases. In summary, the results represent a much more extensive set of data than available from earlier experiments on DNA breakage from ^l25I-decay, and may provide new mechanistic insights

[en] Full text: The mechanism by which charges migrate in DNA continues to be an area of active fundamental research. We have used bibenzimidazole ligands, which bind tightly to the minor groove of DNA, to study the migration of electron deficient centers produced by abstraction of an electron from DNA bases, using the pulse radiolysis technique, in aqueous solution at ambient temperature. Formation of one-electron oxidized ligands by migration of the hole is monitored by time-resolved spectrophotometry, at various ligand/DNA base ratios. These studies build on our previous work with methylpromine and Hoechst 33342 to include a series of ligands which possess both electron donating and withdrawing substituents on the terminal benzene ring. By establishing redox equilibria with promethazine, which binds only weakly to DNA, it has been possible to measure the one-electron reduction potentials, E(1)R, of the DNA-bound ligand radicals for comparison with E(1)R values for the unbound ligands. In all cases E(1)R values are found to increase upon binding of the ligands to DNA. The extent of hole migration along DNA is found to correlate with the rate of electron transfer, a process which is activation energy-controlled

[en] Full text: The highly localised radiochemical damage arising from the decay of Auger electron emitting isotopes such as ¹²⁵I is well known, as is the potential to exploit this phenomenon in cancer therapy, by targeting the effect to the DNA of tumour cells. Most studies have focussed on incorporating the isotope into DNA, for example by using a labelled DNA precursor such as ^l25I-iododeoxyuridine. On average, each decay of ¹²⁵I in DNA results in DNA double-stranded breaks, and only 30-100 such events correspond to a lethal lesion. Detailed analysis of the DNA strand breakage in ¹²⁵I-oligoDNA have shown that the DNA strand containing the covalently attached ¹²⁵I sustains more damage than the 'opposite' strand. There are some limitations to the use of precursors such as ¹²⁵I-iododeoxyuridine, and ^l25I-labeled DNA ligands represent an alternative way to target the Auger emitting isotope to DNA. However, the lower level of damage in the unlabelled strand of ¹²⁵I-DNA, suggests that the cytotoxic potency of ¹²⁵I-labeled DNA ligands may be somewhat less than that of ¹²⁵I-decays in DNA labelled with ¹²⁵-iododeoxyuridine. Accordingly, the cytotoxicity of ¹²⁵I-labeled iodoHoechst 33258 has been investigated in K562 cells, by clonogenic survival in soft agar. Incubation of cells in 5 μM ¹²⁵I-iodoHoechst 33258 and subsequent measurement of l25I in washed cells established that 50% of the drug was taken-up by the cells. Similarly, measurement of ¹²⁵I in washed nuclei prepared by treatment with neutral detergent indicated that 60% of the cellular drug was in the nucleus. ¹²⁵I decays were accumulated in the labeled cells for extended periods at -70 deg C in 10% DMSO, or for shorter periods at 0.3 deg C , ±DMSO. These storage conditions compromise the clonogenic survival of control (no ¹²⁵I-ligand ) cells. Compared to cells not exposed to the cold, which have a cloning efficiency of about 55%, it was reduced to 32% and 12% for cells stored at -70 deg C for 48 hours and 96 hours, respectively. Similarly, storage at 0.3 deg C for 8 hours and 48 hours reduced cloning efficiency to 20% and 8% in the absence of DMSO. Cloning efficiency was reduced to 36% and 22% after storage at 0.3 deg C for 8 hours and 48 hours, respectively, in the presence of 10% DMSO. Using the relevant cloning controls, survival curves were obtained in which the dose was expressed as ¹²⁵I decays per cell. From this data, D₃₇ values of 200 decays per cell, 150 decays per cell and 115 decays per cell were obtained for the frozen and 0.3 deg C conditions, with and without DMSO, respectively

[en] Full text: Exposure of cultured cells to an internal source of ionising radiation, such as a radioactive isotope, differs substantially from external irradiation in the determination of delivered dose. In some cases, the radioactive isotope cannot be quickly and completely removed from cells before plating for clonogenic survival assay. This provides an additional dose of irradiation which is not easy to calculate. The contribution of this phenomenon to the cell survival is especially important if a radioactive isotope is incorporated into DNA, or a DNA-binding ligand is labelled with the isotope. The correction of the cell survival due to additional dose cannot be calculated using a simple analytical expression, since the isotope is present in the cells during colony growth. We have developed a Monte Carlo model which simulates the process of the colony growth, and takes into account the extent of damage from isotope decays accumulated between consequent cell divisions. The model considers such factors as cell cycle time, radiosensitivity, colony growth inhibition, isotope specific (per cell) activity, partition of isotope in daughter cells, isotope half-life time, isotope efflux. The model allows estimation of the impact of the irradiation during colony formation on the distribution of colony size, and on the calculation of the survival correction factor, which depends mainly on the isotope cell-specific activity. We applied the model to interpret the difference in survival of K652 cells exposed to ¹²⁵I decays with various cell-specific activities: 0.45, 3.21 and 7.42 decays/cell/hour. The cells were treated with ¹²⁵I - labelled Hoechst 33258 which binds to DNA in cell nucleus. After accumulation of ¹²⁵I decays under non-growth conditions, cells were plated for clonogenic survival assay. The survival correction factors calculated from the model for the given values of ¹²⁵I cell-specific activity are in good correlation with differences between experimental survival curves. More generally, the simulations show that the persistence of the isotope during colony development does not obviate the use of the colony scoring method

[en] Full text: Decay of Auger electron emitting isotopes such as ¹²³I and ¹²⁵I results in high local energy deposition. When such an event occurs in association with DNA it induces cytotoxic DNA damage that makes it possible to exploit Auger-emitters in radioimmunotherapy. The efficiency of induction of cytotoxic lesions by decay of DNA-associated ¹²⁵I, the prototype Auger-emitter, is well established, but its long half-life (60 days) is a limitation. A much shorter half-life (13.2 hours) of another Auger emitting iodine isotope, ¹²³I is an obvious advantage. However decay of ¹²³I generates an average of 8 - 11 Auger electrons compared to about 15 - 21 electrons for ¹²⁵I, so the efficiency of DNA damage and subsequent cytotoxicity might be somewhat lower for ¹²³I. We investigated the biological consequences of decay of DNA-associated ¹²³I using breakage of plasmid DNA and clonogenic survival of cultured cells following incubation with DNA binding ligand ¹²³I-iodoHoechst 33258, as endpoints. The efficiency of double strand break induction in pBR 322 plasmid by decay of ¹²³I was 0.62, compared to 0.82 per decay of ¹²⁵I in the same experimental system. In the presence of dimethylsulfoxide, the values were 0.54 and 0.65 for decay of ¹²³I and ¹²'5I respectively. Clonogenic survival studies with K562 cells demonstrated that about 370 - 430 decays of ¹²³I per cell constitute a lethal event, compared to130-140 decays of ¹²⁵I per cell. In considering the possible exploitation of the Auger effect in cancer therapy, the modest decrease in DNA breakage and cytotoxic efficiency of ¹²³I might be compensated for by the advantages of the much shorter half life

[en] 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. (orig.)

[en] Full text: Methylproamine is the current lead compound of a new class of DNA-binding radioprotectors being developed in the Research Laboratories at the Peter MacCallum Cancer Centre in Melbourne. The salient features of methylproamine are its radioprotective potency and the generic nature of the apparent radioprotective mechanism. Like the 'classic' aminothiol radioprotectors exemplified by amifostine, methylproamine suppresses the initial radiochemical damage induced in DNA by ionising radiation. However, survival curve studies with cultured cells have demonstrated that methylproamine is 100-fold more potent than WR1065, the active metabolite of amifostine. The radioprotective mechanism seems to involve reduction by the DNA-bound drug of transient radiation-induced oxidising species on DNA. This mechanism implies some electron transfer along DNA, from the DNA-bound drug to the oxidising species. In vivo radioprotection of mouse lung, GI tract and bone marrow has been demonstrated following systemic administration of methylproamine to mice. The commercial potential of radioprotectors resides in two distinct arenas. Until recently, most of our efforts have focused on the use of methylproamine to protect normal tissues in cancer radiotherapy patients, but a quite different opportunity arises from the imperative to develop countermeasures to the threat of radiation terrorism. Our prospects in both arenas have been lifted by the results of synthesis and screening of a pilot library of ∼50 methylproamine analogues, promising the emergence of new lead drugs. In particular, although methylproamine is a potent radioprotector, at higher concentrations it becomes cytotoxic, but one member of the pilot library shows a wider efficacy 'window'. We plan to continue this lead optimisation process by synthesis and screening of a much larger library of analogues, and we are seeking the support of a commercial partner