Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for anti-gene radiotherapy.

Targeting of radiation damage to specific DNA sequences is the essence of antigene radiotherapy. This technique also provides a tool to study molecular mechanisms of DNA repair on a defined, single radiodamaged site. We achieved such sequence-specific radiodamage by combining the highly localized DNA damage produced by the decay of Auger-electron-emitters such as 125I with the sequence-specific action of triplex-forming oligonucleotides (TFO). TFO complementary to polypurine-polypyrimidine regions of human genes were synthesized and labeled with 125I-dCTP by the primer extension method. 125I-TFO were delivered into cells with several delivery systems. In addition, human enzymes capable of supporting DNA single-strand-break repair were isolated and assessed for their role in the repair of this lesion. Also, the mutagenicity and repairability of 125I-TFO-induced double strand breaks (DSB) were assessed by repair of a plasmid possessing a site-specific DSB lesion. Using plasmids containing target polypurine-polypyrimidine tracts, we obtained the fine structure of sequence-specific DNA breaks produced by decay of 125I with single-nucleotide resolution. We showed that the designed 125I-TFO in nanomolar concentrations could bind to and introduce double-strand breaks into the target sequences in situ, i.e., within isolated nuclei and intact digitonin-permeabilized cells. We also showed 125I-TFO-induced DSB to be highly mutagenic lesions resulting in a mutation frequency of nearly 80%, with deletions comprising the majority of mutations. The results obtained demonstrate the ability of 125I-TFO to target specific sequences in their natural environment--within eucaryotic nucleus. Repair of 125I-TFO-induced DNA damage should typically result in mutagenic gene inactivation.     

Characterization of a complex 125I-induced DNA double-strand break: implications for repair

PURPOSE: To examine the role of radiation-induced DNA double-strand break (DSB) structural organization in DSB repair, and characterize the structural features of 125I-induced DSBs that may impact the repair process. METHODS: Plasmid DNA was linearized by sequence-specific targeting using an 125I-labeled triplex-forming oligonucleotide (TFO). Following isolation from agarose gels, base damage structures associated with the DSB ends in plasmids linearized by the 125I-TFO were characterized by probing with the E. coli DNA damage-specific endonuclease and DNA-glycosylases, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-glycosylase (Fpg). RESULTS: Plasmid DNA containing DSBs produced by the high-LET-like effects of 125I-TFO has been shown to support at least 2-fold lower end joining than gamma-ray linearized plasmid, and this may be a consequence of the highly complex structure expected near an 125I-induced DSB end. Therefore, to determine if a high density of base damage exists proximal to the DSBs produced by 125I-TFOs, short fragments of DNA recovered from the DSB end of 125I-TFO-linearized plasmid were enzymatically probed. Base damage and AP site clustering was demonstrated within 3 bases downstream and 7 bases upstream of the targeted base. Furthermore, the pattern and extent of base damage varied depending upon the presence or absence of 2 M DMSO during irradiation. CONCLUSIONS: 125I-TFO-induced DSBs exhibit a high degree of base damage clustering proximal to the DSB end. At least 60% of the nucleotides within 10 bp of the 125I decay site are sensitive to cleavage by endo IV, endo III, or Fpg following damage accumulation in the presence of DMSO, whereas > or = 80% are sensitive in the absence of DMSO. The high degree of base damage clustering associated with the 125I-TFO-induced DSB end may be a major factor leading to its negligible in vitro repair by the non-homologous end-joining pathway (NHEJ).     

On the biological efficiency of I-123 and I-125 decay on the molecular level

Purpose: To evaluate DNA damage of Auger emitters by numerical modelling at the molecular level.

Material and methods: Energy emission spectra of I-123 and I-125 were used as input data for a computer code that simulates the complete transport of electrons and photons from the physical stage up to the primary chemical stage at 10^?7 s. The simulation was performed in a complex environment of liquid water, DNA structures and scavengers. Electron and photon interactions with the DNA molecules were carefully managed. Simulations were carried out with both I-123 and I-125 bound to a pBR322 plasmid or free in its vicinity.

Results: The distributions of direct and indirect single strand breaks (SSB) and double strand breaks (DSB) as a function of the kinetic energy of the emitted Auger electrons show that damage is caused primarily by electrons with energies lower than 800 eV, while higher energy electrons are mainly involved in indirect effects. The yields per unit energy emitted strengthen this fact. When compared to experimental values, the calculated yields of linearization (LE) and relaxation (RE) events show good agreement as well as does the ratio LE/RE for each radionuclide and the ratio I-125/I-123 in the case of LE.     

Calculated DNA Damage from Gadolinium Auger Electrons and Relation to Dose Distributions in a Head Phantom

Purpose: To calculate the number of ¹⁵⁷Gadolinium (¹⁵⁷Gd) neutron capture induced DNA double strand breaks (DSB) in tumor cells resulting from epithermal neutron irradiation of a human head when the peak tissue dose is 10?Gy. To assess the lethality of these Gd induced DSB.

Materials and Methods: DNA single and double strand breaks from Auger electrons emitted during ¹⁵⁷Gd(n,gamma) events were calculated using an atomistic model of B‐DNA with higher‐order structure. When combined with gadolinium neutron capture reaction rates and neutron and photon physical dose rates calculated from the radiation transport through a model of the human head with explicit tumors, peak tissue dose can be related to the number of Auger electron induced DSB in tumor cell DNA. The lethality of these DNA DSB were assessed through a comparison with incorporated ¹²⁵I decay cell survival curves and second comparison with the number of DSB resulting from neutron and photon interactions.

Results: These calculations on a molecular scale (microscopic calculations) indicate that for incorporated ¹⁵⁷Gd, each neutron capture reaction results in an average of 1.56±0.16 DNA single strand breaks (SSB) and 0.21±0.04 DBS in the immediate vicinity (～40?nm) of the neutron capture. In an example case of Gd Neutron Capture Therapy (GdNCT), a 1?cm radius midline tumor, peak normal tissue dose of 10?Gy, and a tumor concentration of 1000?ppm Gd, result in a maximum of 140±27 DSBs per tumor cell.

Conclusions: The number of DSB from the background radiation components is one order of magnitude lower than the Gd Auger electron induced DSB. The cell survival of mammalian cell lines with a similar amount of complex DSB induced from incorporated ¹²⁵I decay yield one to two magnitudes of cell killing. These two points indicate that gadolinium auger electrons could significantly contribute to cell killing in GdNCT.     

Formation and repair of clustered damaged DNA sites in high LET irradiated cells

Purpose: Radiation with high linear energy transfer (LET) produces clustering of DNA double-strand breaks (DSB) as well as non-DSB lesions. Heat-labile sites (HLS) are non-DSB lesions in irradiated cells that may convert into DSB at elevated temperature during preparation of naked DNA for electrophoretic assays and here we studied the initial formation and repair of these clustered damaged sites after irradiation with high LET ions.

Materials and methods: Induction and repair of DSB were studied in normal human skin fibroblast (GM5758) after irradiation with accelerated carbon and nitrogen ions at an LET of 125 eV/nm. DNA fragmentation was analyzed by pulsed-field gel electrophoresis (PFGE) and by varying the lysis condition we could differentiate between prompt DSB and heat-released DSB.

Results: Before repair (t = 0 h), the 125 eV/nm ions produced a significant fraction of heat-released DSB, which appeared clustered on DNA fragments with sizes of 1 Mbp or less. These heat-released DSB increased the total number of DSB by 30–40%. This increase is similar to what has been found in low-LET irradiated cells, suggesting that the relative biological effectiveness (RBE) for DSB induction will not be largely affected by the lysis temperature. After 1–2 hours repair, a large fraction of DSB was still unrejoined but there was essentially no heat-released DSB present.

Conclusions: These results suggest that high LET radiation, as low LET gamma radiation, induces a significant fraction of heat-labile sites which can be converted into DSB, and these heat-released DSB may affect both induction yields and estimates of repair.     

DNA double-strand breaks induced by decay of 123I-labeled Hoechst 33342: Role of DNA topology

Purpose: To determine double-strand-break (DSB) yields produced by decay of minor-groove-bound ¹²³I-labeled Hoechst 33342 (¹²³IEH) in supercoiled (SC) and linear (L) forms of pUC19 DNA, to compare strand-break efficiency of ¹²³IEH with that of ¹²⁵IEH, and to examine the role of DNA topology in DSB induction by these Auger electron emitters.

Materials and methods: Tritium-labeled SC and L pUC19 DNA were incubated with ¹²³IEH (0–10.9 MBq) at 4°C. After ¹²³I had completely decayed (10 days), samples were analyzed on agarose gel, and single-strand-break (SSB) and DSB yields were measured.

Results: Each ¹²³I decay in SC DNA produces a DSB yield of 0.18 ± 0.01. On the basis of DSB yields for ¹²⁵IEH (0.52 ± 0.02 for SC and 1.62 ± 0.07 for L, reported previously) and dosimetric expectations, a DSB yield of ～0.5 (3 × 0.18) per ¹²³I decay is expected for L DNA. However, no DSB are observed for the L form, even after ～2 × 10¹¹ decays of ¹²³I per μg DNA, whereas a similar number of ¹²⁵I decays produces DSB in ～40% of L DNA.

Conclusion: ¹²³IEH-induced DSB yield for SC but not L DNA is consistent with the dosimetric expectations for Auger electron emitters. These studies highlight the role of DNA topology in DSB production by Auger emitters and underscore the failure of current theoretical dosimetric methods per se to predict the magnitude of DSB.     

Assessment of DNA damage produced by 125I‐triplex‐forming oligonucleotides in cells

Purpose: Triplex‐forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger‐electron‐emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two ¹²⁵I‐labeled TFOs, one with a single target in the genome and another with multiple targets.

Materials and methods: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone γ‐H2AX foci formed at the sites of DNA double strand breaks (DSBs).

Results: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more γ‐H2AX foci per cell than the TFO with a single target.

Conclusion: Since the two methods gave comparable results, measuring the number of γ‐H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.     

Cytotoxicity of cigarette smoke condensate is not due to DNA double strand breaks: Comparative studies using radiosensitive mutant and wild-type CHO cells

Purpose:?To determine whether cigarette smoke condensate (CSC) without metabolic activation induces direct DNA double strand breaks (DSB) in the G1 phase of various radiosensitive mutants of CHO cells and whether these breaks display collateral hypersensitivity to CSC with respect to cell killing.

Materials & methods:?We treated the G1-phase cultures of wild-type and DNA repair deficient mutants of CHO cells with various concentrations of CSC and examined the cell survival by colony formation assay and the induction of DNA double strand breaks by constant field gel electrophoresis as well as the phophorylated histone H2-A variant X (γ-H2AX) assay.

Results:?Gel analysis and γ-H2AX focus assay showed significantly fewer, but still detectable levels of DSB per cell after CSC treatment compared to ionizing radiation (IR) exposures, even when equitoxic radiation exposures were delivered at a low dose rate over the same 8-hour exposure used for CSC treatments. None of the three non-homologous end joining (NHEJ) deficient mutants were remarkably hypersensitive to CSC compared to wild-type cells. In contrast, UV-1 cells that are hypersensitive to several base damage and cross-linking agents showed a higher sensitivity to CSC compared to the other CHO cell lines.

Conclusions:?DNA DSB produced directly by CSC are not principally responsible for its cytotoxicity. Further, the present study does not rule out the possibility that some of these lesions may secondarily result in DSB, such as may occur during impeded DNA replication and whose repair may require systems other than NHEJ.     

Plasmid breakage by 125I-labelled DNA ligands: Effect of DNA-iodine atom distance on breakage efficiency

Purpose: The aim of the study is to establish the relationship between the efficiency of DNA double-stranded breakage by ¹²⁵I-labelled DNA ligands and the distance from the decaying atom to the helical axis.

Materials and methods: Two new iodinated minor groove binding ligands were synthesized which, on the basis of molecular modelling studies, place the iodine atom at different distances from the DNA helical axis (namely 7.4 and 11.2 A°). Plasmid DNA breakage experiments, in both buffer-only and buffer + 2M dimethylsulfoxide (DMSO), were used to determine the efficiency of induction of internal double-stranded breaks (DSB) of the two new ligands, as well as that for ¹²⁵I-Hoechst 33258, which is characterized by a helical axis-iodine atom distance of 9.1 A°.

Results: The results showed a progressive decrease in the efficiency of DNA DSB induction with the axis-iodine atom distance, for both incubation conditions. The distance-damage relationship was somewhat steeper than previously predicted from the theoretical studies by Humm and Charlton, based on radical-mediated damage. Another distinctive trend was revealed by comparison of breakage efficiency with and without DMSO. The extent of DMSO protection increased significantly with DNA-iodine distance.

Conclusions: The steeper than predicted decrease in DSB induction with DNA-iodine distance is consistent with a substantial contribution to DNA breakage of the charge neutralization effect (arising from the transient positive charge left on the daughter Te atom), and the expectation that this contribution would be very dependent on the distance of the site of hole injection from the base-pair π-stack. An important caveat to the results and conclusions is the need to confirm the estimated helical axis-iodine distances with X-ray crystallography studies, and for further exemplification with a more extensive collection of DNA ligands.     

Effect of distance between decaying 125I and DNA on Auger-electron induced double-strand break yield

Abstract

Purpose: To determine the possible effects of ¹²⁵I-to-DNA distance on the magnitude and mechanism of Auger-electron induced-double-strand break (DSB) production.

Materials and methods: We have synthesized a series of ¹²⁵I-labeled Hoechst (H) derivatives (¹²⁵IE–H, ¹²⁵IB–H, ¹²⁵I-C₈–H and ¹²⁵I-C₁₂–H). While all four molecules share a common DNA minor groove binding bis-benzimidazole motif, they are designed to position ¹²⁵I at varying distances from the DNA helix. Each Hoechst derivative was incubated at 4°C in phosphate buffered saline (PBS) together with supercoiled (SC) ³H-pUC19 plasmid DNA (ratio 3:1) ± the ?OH scavenger dimethyl sulfoxide (DMSO) (0.2 M). Aliquots were analyzed on agarose gels over time and DSB yields per decay of ¹²⁵I atom were determined. Docking of the iodinated compounds on a DNA molecule was carried out to determine the distance between the iodine atom and the central axis of DNA.

Results: In the absence of DMSO, the results show that the DSB yields decrease monotonically as the ¹²⁵I atom is distanced – by 10.5 Å to 13.9 Å – from the DNA helix (¹²⁵IEH: 0.52 ± 0.01; ¹²⁵IB–H: 0.24 ± 0.03; ¹²⁵I-C₈–H: 0.18 ± 0.02; ¹²⁵I-C₁₂–H: 0.10 ± 0.00). In the presence of DMSO, DSB yields for ¹²⁵IEH (0.49 ± 0.02) and ¹²⁵IB-H (0.26 ± 0.04) remain largely unchanged indicating that DSB are entirely produced by direct effects. Strikingly, ¹²⁵I-C₈–H or ¹²⁵I-C₁₂–H, did not produce detectable DSB in the presence of DMSO under similar conditions suggesting when ¹²⁵I atom is positioned >?12 Å from the DNA, DSB are entirely produced by indirect effects.

Conclusion: These results suggest that at a critical distance between the ¹²⁵I atom and the DNA helix, DSB production switches from an ‘all’ direct to an ‘all’ indirect mechanism, the latter situation being comparable to the decay of ¹²⁵I free in solution. These experimental findings were correlated with theoretical expectations based on microdosimetry.     

Modelling of cell killing due to sparsely ionizing radiation in normoxic and hypoxic conditions and an extension to high LET radiation

Abstract

Purpose: An approach for describing cell killing with sparsely ionizing radiation in normoxic and hypoxic conditions based on the initial number of randomly distributed DNA double-strand breaks (DSB) is proposed. An extension of the model to high linear energy transfer (LET) radiation is also presented.

Materials and methods: The model is based on the probabilities that a given DNA giant loop has one DSB or at least two DSB. A linear combination of these two classes of damage gives the mean number of lethal lesions. When coupled with a proper modelling of the spatial distribution of DSB from ion tracks, the formalism can be used to predict cell response to high LET radiation in aerobic conditions.

Results: Survival data for sparsely ionizing radiation of cell lines in normoxic/hypoxic conditions were satisfactorily fitted with the proposed parametrization. It is shown that for dose ranges up to about 10 Gy, the model describes tested experimental survival data as good as the linear-quadratic model does. The high LET extension yields a reasonable agreement with data in aerobic conditions.

Conclusions: A new survival model has been introduced that is able to describe the most relevant features of cellular dose-response postulating two damage classes.     

Influence of DNA double‐strand break rejoining on clonogenic survival and micronucleus yield in human cell lines

Purpose: To examine the role of DNA double‐strand break (DSB) rejoining in cell survival and micronucleus yield after ⁶⁰Co γ‐irradiation.

Materials and methods: Thirteen human cell lines (six glioblastoma, five prostate, one melanoma, one squamous cell carcinoma) were irradiated with ⁶⁰Co γ‐rays to doses of 0–10?Gy for cell survival and micronucleus measurements and 0–100?Gy for DSB rejoining. Measurements were performed using standard clonogenic, micronucleus and constant‐field gel electrophoresis assays.

Results: Radioresistance and micronucleus yield were positively correlated (r=0.74, p=0.004). A significant cell type‐dependent correlation was demonstrated between total (0–20?h) DSB rejoining and cell survival (r=0.86, p=0.03 for glioblastomas; r=0.79, p=0.04 for other cell lines), with more resistant cell lines showing higher levels of DSB rejoining. No relationship was apparent between fast (0–2?h) or slow (2–20?h) DSB rejoining and clonogenic survival. While there was no relationship between total or slow DSB rejoining and micronucleus yield, a significant and cell type‐specific correlation emerged between fast rejoining and micronucleus yield for the glioblastomas (r=0.89, p=0.04) and other cell lines (r=0.76, p=0.04). Cell lines with higher levels of DSB rejoining within 2?h of irradiation showed higher yields of micronuclei.

Conclusion: Fast DSB rejoining, possibly through interaction with slow DSB rejoining, appears to play an important role in the formation of micronuclei. However, total DSB rejoining reflects intrinsic radiosensitivity. Consideration of differences in DSB rejoining kinetics might contribute to a better understanding of the significance of cell survival and micronucleus data in the clinical and radiation protection setting.     

Radiation-induced genomic instability in repair deficient mutants of Chinese hamster cells

Purpose: To determine the role of single (SSB) and double strand break (DSB) repair in the induction and propagation of radiation-induced instability.

Materials and methods: Two defined hamster cell lines with known DNA repair deficiencies in DSB repair (XR-C1) and base excision repair (EM-C11) and the parental wild-type line (CHO-9) were used. The rate of micronucleus formation, apoptosis and survival were measured at 0, 7 and 14 days after X-ray radiation.

Results: An enhanced rate of production of damaged cells was observed in wild type and the repair deficient mutants after irradiation. This was cell type, dose and time-dependent. All cells demonstrated delayed death up to day 14 after irradiation along with an elevated apoptosis frequency. The yield of micronuclei was not significantly increased in the wild-type cells, but was in the mutant cells, over the dose and time range studied. For all three endpoints the increase in damage was most pronounced in the SSB deficient cell line.

Conclusions: SSB and/or oxidized base damage play a major role, rather than DSB, in radiation induced instability.     

Activation of diverse pathways to apoptosis by 125IdUrd and γ‐photon exposure

Purpose: To delineate the mechanisms underlying induction of apoptosis in malignant cells irradiated by DNA‐incorporated iodine‐125 or γ‐photons.

Materials and methods: Human tumor cells (RKO, LS174T, TE671, and MCF7) were irradiated by DNA‐incorporated 5‐[¹²⁵I]iodo‐2′‐deoxyuridine (¹²⁵IdUrd) or by γ‐photons. Clonogenic survival was determined by the colony‐forming assay. Caspase‐3 induction was measured with a fluorogenic substrate assay, and DNA fragmentation was determined by ligation‐mediated polymerase chain reaction. DNA arrays were used to assess the expression of the B‐cell lymphoma/leukaemia‐2 (Bcl‐2) family and related genes in RKO cells and in caspase‐3‐gene‐defective MCF7 cells.

Results: After ¹²⁵IdUrd or γ‐photon exposure, the highest induction of caspase‐3 was observed in the radiation‐sensitive cell lines (RKO and LS174T). DNA fragmentation was prominent in the radiosensitive cells and undetectable in TE671 (¹²⁵IdUrd and γ‐photons) and MCF7 (¹²⁵IdUrd only) cells. Exposure of RKO and MCF7 cells to ¹²⁵I decay led to up‐regulation of several pro‐apoptotic and anti‐apoptotic Bcl‐2 family genes whereas γ‐irradiation produced minimal activation.

Conclusions: Apoptosis generated by a DNA‐incorporated Auger electron emitter is induced through the mitochondrial/caspase‐3‐mediated pathway and correlates with cellular radiosensitivity. Apoptosis caused by γ‐radiation can be signaled without activation of Bcl‐2 family genes, and DNA fragmentation occurs with or without caspase‐3 activation.     

Calculated strand breaks from 125I in coiled DNA

Purpose: DNA single strand breaks (SSB) and double-strand breaks (DSB) induced by Auger electrons from incorporated ¹²⁵I decay were calculated using a B-DNA model to assess contributions from direct and OH damage and effects of higher-order structure. Three decay sites, linker DNA, nucleosome, and two adjacent nucleosomes, were assessed and compared to experimental data.

Method: A Monte Carlo track structure code for electron was used to track electrons, OH and H radicals through linear and a higher-order model of B-DNA. Direct and indirect DNA hits were scored and used to determine SSB and DSB.

Results: The three different ¹²⁵I decay locations produced different number of DSBs and fraction of radical damage. The average number of DSB per ¹²⁵I decay was 0.83, 0.86 and 1.33, respectively, for the three sites. OH radical attack contributed to or exclusively caused 70%, 57%, and 50%, of the DSBs located in the entire model. When only 10 base pairs on either side of the incorporation site were considered, radical damage contributions were 40%, 25% and 67%, respectively. Locations distant from the site of incorporation, however, consistently yielded 70–80% of the DSB from radical attack.

Conclusions: Coiling of DNA can greatly change both the absolute number of DSB per incorporated ¹²⁵I decay and the relative contributions of radical damage to the local site of decay and, to a lesser extent, the average over all DNA. Higher order structure only slightly affects the number and quality of DNA damage to distant locations, which is mostly from radical attack.     

Antisense radiotherapy: targeting full‐size mdr1 mRNA with 125I‐labelled oligonucleotides

Purpose: Antisense radiotherapy is an approach based on the targeting of mRNA of specific genes by complementary oligonucleotide probes labelled with an Auger‐electron‐emitting radioisotope. Decay of the Auger emitter should specifically destroy the targeted mRNA while producing minimal damage to the rest of mRNA pool and the nuclear DNA. The feasibility of this approach was investigated by using full‐length human multidrug‐resistance gene (mdr1) mRNA as a target.

Materials and methods: Antisense oligonucleotides were labelled with [¹²⁵I] I‐dCTP by primer extension and annealed to target mRNA. Breaks in the target mRNA were analysed by denaturing polyacrylamide gel electriphoresis.

Results: The efficiency of ¹²⁵I‐labelled antisense oligonucleotides in producing RNA strand breaks was tested on short synthetic RNA and DNA targets. The position and specificity of ¹²⁵I‐induced breaks in the full‐length mRNA were then tested and compared with the cleavage of the target by RNase H. The distribution of the breaks in the longer mRNA is different from that in the short RNA targets, most likely due to a complex folding of RNA strands in the full‐length mRNA.

Conclusions: The authors posit that ¹²⁵I‐labelled antisense probes could be useful not only for targeting mRNA, but also as probes for mRNA folding in vivo.     

Kinetics and dose-response of residual 53BP1/γ-H2AX foci: Co-localization,relationship with DSB repair and clonogenic survival

Purpose: Recent studies revealed that some foci produced by phosphorylated histone 2A family member X (γ-H2AX) and tumor suppressor p53 binding protein 1 (53BP1) that co-localize with radiation-induced DNA double-strand breaks (DSB) remain in cells at relatively long times after irradiation and indicated a possible correlation between cellular radiosensitivity and residual foci. In this study, we investigated dose-responses and kinetics for radiation-induced 53BP1/γ-H2AX foci formation in relation to their co-localization, DSB repair and cell survival.

Materials and methods: Cell survival, DSB and foci were analyzed by clonogenic assay, pulsed field gel electrophoresis (PFGE), and confocal laser microscopy, respectively, in normal human fibroblasts (VH-10) and in a cancer cell line (HeLa). Computer analysis was used to determine both the number and the area of foci.

Results: We show that even at doses down to 1 cGy a statistically significant induction of 53BP1 foci is observed. While the number of foci was found to constantly decrease with post-irradiation time, the per-cell normalized area of foci does not change within a time window of approximately 4 h post-irradiation. Co-localization of γ-H2AX and 53BP1 foci is shown to depend on dose and post-irradiation time. No clear correlations were established between radiosensitivity and foci formation because the dose response for 53BP1/γ-H2AX foci may depend on time after irradiation and duration of the cell cycle. We show that the kinetics of foci disappearance within 24 h post-irradiation do not coincide with those of DSB repair.

Conclusions: The data suggest that the post-irradiation time used for estimation of radiosensitivity at therapeutically relevant low doses (e.g., <3 Gy) in proliferating cells by scoring residual foci should be limited by the duration of the cell cycle, and that direct comparison of the kinetics of DSB repair and disappearance of DSB-co-localizing foci is not possible. Therefore, results obtained from the counting of foci should be interpreted with caution in terms of DSB repair.     

Efficiencies of Induction of DNA Double Strand Breaks in Solution by Photoabsorption at Phosphorus and Platinum

Purpose: This paper aims at determining and comparing the cross sections and quantum yields for DNA strand break induction by the Auger effect at the K‐shell of phosphorus and at the L_III‐shell of platinum.

Materials and methods: Using synchrotron radiation, free and Pt‐bound pBR322 plasmid DNA were irradiated in solution with monochromatic X‐rays, the energies of which were 2.153 and 2.147?keV, corresponding to “on” and “below” the phosphorus K‐shell photoabsorption, and 11.566 and 11.542?keV for “above” and “below” the L_III‐shell photoabsorption of platinum, respectively. To suppress indirect effects by hydroxyl radicals, DMSO (1M) was used as a scavenger.

Results: The inner‐shell photoabsorption of phosphorus and of platinum significantly increased the induction of DNA double strand breaks (DSB), whereas it had little effect on single strand break (SSB) induction. The quantum yields for the induction of DSB were calculated to be 0.017 and 1.13, in the case of phosphorus and platinum, respectively.

Conclusions: The value of the quantum yield for the DSB induction of platinum was about 66‐fold larger than that for the phosphorus. These results clearly demonstrate that the quantum yield of DSB depends upon the magnitude of the Auger cascade.     

Time and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity

Purpose:?Proper detection of DNA damage and signal transduction to other proteins following irradiation (IR) is essential for cellular integrity. The serine 15 (Ser15) on p53 is crucial for p53 stabilization and a requirement for transient and permanent cell cycle arrest. Here, we sought to determine the relationship between p53 serine 15 phosphorylation (p53-p-Ser15) on cellular sensitivity and if this modification is associated with DNA double-strand break (DSB) repair.

Materials and methods:?Eight lymphoblastoid cell lines including ataxia-telangiectasia (A-T), Nijmegen breakage syndrome (NBS) and radiosensitive patient derived cell lines were irradiated with 1 Gy, 2 Gy and 5 Gy. Then growth inhibition, p53 induction and phosphorylation on Ser15 as assessed by immunoblotting and DNA DSB repair as assessed by constant field gel electrophoresis were examined.

Results:?Phosphorylation of p53 at Ser15 in control cells rapidly increased, peaking at 3 – 6 hours and then sustained a low level of phosphorylation for up to 6 days following IR. For these cell lines, the amount of p53-p-Ser15 corresponded to the sensitivity of cells and the amount of DNA DSB. In A-T cells, p53-p-Ser15 was reduced in spite of increased DNA DSB. NBS cells had similar phosphorylation dynamics as the control cell line, which was not consistent with their increased sensitivity. Radiosensitive patients' cell lines differed only slightly from controls.

Conclusions:?Cells that are competent in signal transduction have p53-p-Ser15 kinetics corresponding to cellular radiosensitivity as assessed by clonogenicity and DNA DSB repair, and cells impaired in signal transduction lack this correspondence. Therefore, using p53-p-Ser15 as a general marker of radiation sensitivity has confounding factors which may impair proper radiosensitivity prediction.