DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

<p>Abstract</p> <p>Background</p> <p>Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. ¹⁰B captures neutrons and produces an alpha (⁴He) particle and a recoiled lithium nucleus (⁷Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells.</p> <p>Methods</p> <p>Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity.</p> <p>Results</p> <p>Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity.</p> <p>Conclusion</p> <p>Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR.</p

Development of a dual phantom technique for measuring the fast neutron component of dose in boron neutron capture therapy.

[Purpose]: Research and development of various accelerator-based irradiation systems for boron neutroncapture therapy (BNCT) is underway throughout the world. Many of these systems are nearing or have started clinical trials. Before the start of treatment with BNCT, the relative biological effectiveness (RBE) for the fast neutrons (over 10 keV) incident to the irradiation field must be estimated. Measurements of RBE are typically performed by biological experiments with a phantom. Although the dose deposition due to secondary gamma rays is dominant, the relative contributions of thermal neutrons (below 0.5 eV) and fast neutrons are virtually equivalent under typical irradiationconditions in a water and/or acrylic phantom. Uniform contributions to the dose deposited from thermal and fast neutrons are based in part on relatively inaccurate dose information for fastneutrons. This study sought to improve the accuracy in the dose estimation for fast neutrons by using two phantoms made of different materials in which the dose components can be separated according to differences in the interaction cross sections. The development of a “dual phantom technique” for measuring the fast neutron component of dose is reported. [Methods]: One phantom was filled with pure water. The other phantom was filled with a water solution of lithiumhydroxide (LiOH) capitalizing on the absorbing characteristics of lithium-6 (Li-6) for thermal neutrons.Monte Carlo simulations were used to determine the ideal mixing ratio of Li-6 in LiOH solution.Changes in the depth dose distributions for each respective dose component along the central beam axis were used to assess the LiOH concentration at the 0, 0.001, 0.01, 0.1, 1, and 10 wt. % levels. Simulations were also performed with the phantom filled with 10 wt. % [6]LiOH solution for 95%-enriched Li-6. A phantom was constructed containing 10 wt. % [6]LiOH solution based on the simulation results. Experimental characterization of the depth dose distributions of the neutron andgamma-ray components along the central axis was performed at Heavy Water Neutron IrradiationFacility installed at Kyoto University Reactor using activation foils and thermoluminescent dosimeters, respectively. [Results]: Simulation results demonstrated that the absorbing effect for thermal neutrons occurred when the LiOH concentration was over 1%. The most effective Li-6 concentration was determined to be enriched [6]LiOH with a solubility approaching its upper limit. Experiments confirmed that the thermalneutron flux and secondary gamma-ray dose rate decreased substantially; however, the fastneutron flux and primary gamma-ray dose rate were hardly affected in the 10%-[6]LiOH phantom. It was confirmed that the dose contribution of fast neutrons is improved from approximately 10% in the pure water phantom to approximately 50% in the 10%-[6]LiOH phantom. [Conclusions]: The dual phantom technique using the combination of a pure water phantom and a 10%-[6]LiOH phantom developed in this work provides an effective method for dose estimation of the fast neutroncomponent in BNCT. Improvement in the accuracy achieved with the proposed technique results in improved RBE estimation for biological experiments and clinical practice

Adverse effect of mild temperature hyperthermia combined with hexamethylenetetramine compared to its effect combined with tirapazamine in the treatment of solid tumors

This study aimed to assess the effect on solid tumors of mild temperature hyperthermia (MTH) combined with hexamethylenetetramine (HMTA) or tirapazamine (TPZ). Squamous cell carcinoma (SCC VII) tumor-bearing mice were continuously administered 5-bromo-2′-deoxyuridine (BrdU) to label intratumor proliferating (P) cells. Mice received HMTA or TPZ through intraperitoneal single or subcutaneous continuous administration, with or without MTH (40°C, 60 min), followed or not by γ-ray irradiation or cisplatin treatment. After HMTA or TPZ administration without γ-ray irradiation or cisplatin treatment, immediately after γ-ray irradiation, or 1 h after cisplatin treatment, the response of quiescent (Q) cells was assessed in terms of micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cells was determined based on a comparison with non-BrdU-treated tumors. Without MTH, HMTA and TPZ had a nearly equal radiosensitizing and cisplatin sensitivity-enhancing effect on both total and Q cells. With MTH, radio- and cisplatin-sensitizing effects by HMTA were reduced, particularly in the Q cells. In contrast, the enhancing effects of TPZ were increased, particularly in the Q cells. Continuous administration of HMTA and TPZ resulted in higher radio- and cisplatin-sensitizing effects than intraperitoneal single administration. In terms of tumor cytotoxicity as a whole, including Q cells, the administration of γ-ray irradiation or cisplatin treatment combined with continuous HMTA administration is promising, taking into account the clinical use of HMTA. However, MTH should not be combined with HMTA administration

Detection of γH2AX foci in mouse normal brain and brain tumor after boron neutron capture therapy

AimIn this study, we investigated γH2AX foci as markers of DSBs in normal brain and brain tumor tissue in mouse after BNCT.BackgroundBoron neutron capture therapy (BNCT) is a particle radiation therapy in combination of thermal neutron irradiation and boron compound that specifically accumulates in the tumor. 10B captures neutrons and produces an alpha (4He) particle and a recoiled lithium nucleus (7Li). These particles have the characteristics of extremely high linear energy transfer (LET) radiation and therefore have marked biological effects. High LET radiation causes severe DNA damage, DNA DSBs. As the high LET radiation induces complex DNA double strand breaks (DSBs), large proportions of DSBs are considered to remain unrepaired in comparison with exposure to sparsely ionizing radiation.Materials and methodsWe analyzed the number of γH2AX foci by immunohistochemistry 30[[ce:hsp sp="0.25"/]]min or 24[[ce:hsp sp="0.25"/]]h after neutron irradiation.ResultsIn both normal brain and brain tumor, γH2AX foci induced by 10B(n,α)7Li reaction remained 24[[ce:hsp sp="0.25"/]]h after neutron beam irradiation. In contrast, γH2AX foci produced by γ-ray irradiation at contaminated dose in BNCT disappeared 24[[ce:hsp sp="0.25"/]]h after irradiation in these tissues.ConclusionDSBs produced by 10B(n,α)7Li reaction are supposed to be too complex to repair for cells in normal brain and brain tumor tissue within 24[[ce:hsp sp="0.25"/]]h. These DSBs would be more difficult to repair than those by γ-ray. Excellent anti-tumor effect of BNCT may result from these unrepaired DSBs induced by 10B(n,α)7Li reaction

Usefulness of hexamethylenetetramine in combination with chemotherapy using free and pegylated liposomal doxorubicin in vivo, referring to the effect on quiescent cells

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They received hexamethylenetetramine (HMTA) either once intraperitoneally or continuously subcutaneously together with chemotherapy using intraperitoneally administered free doxorubicin (DXR) or intravenously injected pegylated liposomal doxorubicin (PLD). One hour after the free DXR loading or 24 h after the PLD loading, the response of intratumor quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cell population was determined from the tumors not treated with BrdU. Encapsulation of DXR into pegylated liposomes significantly enhanced cytotoxicity, especially in Q cells. HMTA, especially when administered continuously, efficiently increased the sensitivity to DXR, particularly in Q cells. The increase in sensitivity on the continuous rather than single administration of HMTA was a little clearer in the total cell population than in Q cells. DXR's encapsulation into pegylated liposomes and combination with HMTA, particularly when administered continuously, apparently reduced the difference in sensitivity to free DXR between the total and Q cell populations. In terms of the tumor cell-killing effect as a whole, including Q cells, the encapsulation of DXR into pegylated liposomes and combination with HMTA, particularly through continuous administration, are very promising, taking into account that HMTA has been used clinically

FANCD1/BRCA2 Plays Predominant Role in the Repair of DNA Damage Induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA−/−, FANCC−/−, FANCA−/−C−/−, FANCD2−/− cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG−/− and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ

Dose-rate effect was observed in T98G glioma cells following BNCT.

[Background]It is generally said that low LET radiation produce high dose-rate effect, on the other hand, no significant dose rate effect is observed in high LET radiation. Although high LET radiations are produced in BNCT, little is known about dose-rate effect of BNCT. [Materials and methods]T98G cells, which were tumor cells, were irradiated by neutron mixed beam with BPA. As normal tissue derived cells, Chinese hamster ovary (CHO-K1) cells and DNA double strand breaks (DNA-DSBs) repair deficient cells, xrs5 cells were irradiated by the neutrons (not including BPA). To DNA-DSBs analysis, T98G cells were stained immunochemically with 53BP1 antibody. The number of DNA-DSBs was determined by counting 53BP1 foci. [Results]There was no dose-rate effect in xrs5 cells. D0 difference between 4 cGy/min and 20 cGy/min irradiation were 0.5 and 5.9 at the neutron and gamma-ray irradiation for CHO-K1, and 0.3 at the neutron for T98G cells. D0 difference between 20 cGy/min and 80 cGy/min irradiation for T98G cells were 1.2 and 0.6 at neutron irradiation plus BPA and gamma-ray. The differences between neutron irradiations at the dose rate in T98G cells were supported by not only the cell viability but also 53BP1 foci assay at 24 h following irradiation to monitor DNA-DSBs. [Conclusion]Dose-rate effect of BNCT when T98G cells include 20 ppm BPA was greater than that of gamma-ray irradiation. Moreover, Dose-rate effect of the neutron beam when CHO-K1 cells did not include BPA was less than that of gamma-ray irradiation These present results may suggest the importance of dose-rate effect for more efficient BNCT and the side effect reduction

A Bystander effect observed in boron neutron capture therapy: A study of the induction of mutations in the HPRT locus

To investigate bystander mutagenic effects indued by alpha-particles during boron neutron cupture therapy, we mixed cells that were electroporated with boroncaptate sodium (BSH), which led to the accumulation of B-10 inside the cells, and cells that did not contain the boron compound. The BSH containing cells were irradiated with alpha particles produced by B(j,a)Li reaction, where as cells without boron were affected only by the 1H(n,r)2H and 14N(n,p)14C reactions