Boron concentration measurement in biological tissues by charged particle spectrometry

Measurement of boron concentration in biological tissues is a fundamental aspect of boron neutron capture therapy, because the outcome of the therapy depends on the distribution of boron at a cellular level, besides on its overall concentration. This work describes a measurement technique based on the spectroscopy of the charged particles emitted in the reaction ¹⁰B(n,α)⁷Li induced by thermal neutrons, allowing for a quantitative determination of the boron concentration in the different components that may be simultaneously present in a tissue sample, such as healthy cells, tumor cells and necrotic cells. Thin sections of tissue containing ¹⁰B are cut at low temperatures and irradiated under vacuum in a thermal neutron field. The charged particles arising from the sample during the irradiation are collected by a thin silicon detector, and their spectrum is used to determine boron concentration through relatively easy calculations. The advantages and disadvantages of this technique are here described, and validation of the method using tissue standards with known boron concentrations is presented.     

Detailed dosimetry calculation for in-vitro experiments and its impact on clinical BNCT

PurposeBoron Neutron Capture Therapy (BNCT) is a form of hadrontherapy based on the selective damage caused by the products of neutron capture in ¹⁰B to tumour cells. BNCT dosimetry strongly depends on the parameters of the dose calculation models derived from radiobiological experiments. This works aims at determining an adequate dosimetry for in-vitro experiments involving irradiation of monolayer-cultured cells with photons and BNCT and assessing its impact on clinical settings.M&MDose calculations for rat osteosarcoma UMR-106 and human metastatic melanoma Mel-J cell survival experiments were performed using MCNP, transporting uncharged particles for KERMA determinations, and secondary particles (electrons, protons, ¹⁴C, ⁴He and ⁷Li) to compute absorbed dose in cultures. Dose-survival curves were modified according to the dose correction factors determined from computational studies. New radiobiological parameters of the photon isoeffective dose models for osteosarcoma and metastatic melanoma tumours were obtained. Dosimetry implications considering cutaneous melanoma patients treated in Argentina with BNCT were assessed and discussed.ResultsKERMA values for the monolayer-cultured cells overestimate absorbed doses of radiation components of interest in BNCT. Detailed dose calculations for the osteosarcoma irradiation increased the relative biological effectiveness factor RBE_1% of the neutron component in more than 30%. The analysis based on melanoma cases reveals that the use of survival curves based on KERMA leads to an underestimation of the tumour doses delivered to patients.ConclusionsConsidering detailed dose calculation for in-vitro experiments significantly impact on the prediction of the tumor control in patients. Therefore, proposed methods are clinically relevant.     

Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.     

Chromosome aberrations induced in human peripheral blood lymphocytes using heavy particles from 10 B (n, ) 7 Li reaction

The relationship between α-particle dose and chromosome aberration yield was studied in human peripheral blood leukocytes cultured in vitro. The α-irradiation was produced from thermal neutron capture by boron, according to the nuclear reaction ¹⁰B (n, α)⁷Li. Blood samples containing 49 μg ¹⁰B per ml were exposed to the thermal neutrons in a reactor at a flux density of 2·10⁷n/cm²s. By subtracting the rad dose due to the reactor radiations alone from that due to both boron capture and the reactor radiations, the rad dose rate of heavy particles was estimated. The dicentric yield appeared to follow a linear response up to about 18 rad and then showed signs of “saturation”. Comparison with 250 kV X-ray data (doses up to 510 rad) gave an RBE of 22.97.     

The effectiveness of the high-LET radiations from the boron neutron capture [<Superscript>10</Superscript>B(n,α)<Superscript>7</Superscript>Li] reaction determined for induction of chromosome aberrations and apoptosis in lymphocytes of human blood samples

Provided that a selective accumulation of ¹⁰B-containing compounds is introduced in tumor cells, following irradiation by thermal neutrons produces high-LET alpha-particles (⁴He) and recoiling lithium-7 (⁷Li) nuclei emitted during the capture of thermalized neutrons (0.025 eV) from ¹⁰B. To estimate the biological effectiveness of this boron neutron capture [¹⁰B(n,α)⁷Li] reaction, the chromosome aberration assay and the flow cytometry apoptosis assay were applied. At the presence of the clinically used compounds BSH (sodium borocaptate) and BPA (p-boronophenylalanine), human lymphocytes were irradiated by sub-thermal neutrons. For analyzing chromosome aberrations, human lymphocytes were exposed to thermally equivalent neutron fluences of 1.82 × 10¹¹ cm^?2 or 7.30 × 10¹¹ cm^?2 (corresponding to thermal neutron doses of 0.062 and 0.248 Gy, respectively) in the presence of 0, 10, 20, and 30 ppm of BSH or BPA. Since the kerma coefficient of blood increased by 0.864 × 10^?12 Gy cm² per 10 ppm of ¹⁰B, the kerma coefficients in blood increase from 0.34 × 10^?12 cm² (blood without BSH or BPA) up to 2.93 × 10^?12 Gy cm² in the presence of 30 ppm of ¹⁰B. For the ¹⁰B(n, α)⁷Li reaction, linear dose–response relations for dicentrics with coefficients α = 0.0546 ± 0.0081 Gy^?1 for BSH and α = 0.0654 ± 0.0075 Gy^?1 for BPA were obtained at 0.062 Gy as well as α = 0.0985 ± 0.0284 Gy^?1 for BSH and α = 0.1293 ± 0.0419 Gy^?1 for BPA at 0.248 Gy. At both doses, the corresponding ¹⁰B(n, α)⁷Li reactions from BSH and BPA are not significantly different. A linear dose–response relation for dicentrics also was obtained for the induction of apoptosis by the ¹⁰B(n, α)⁷Li reaction at 0.248 Gy. The linear coefficients α = 0.0249 ± 0.0119 Gy^?1 for BSH and α = 0.0334 ± 0.0064 Gy^?1 for BPA are not significantly different. Independently of the applied thermal neutron doses of 0.062 Gy or 0.248 Gy, the ¹⁰B(n, α)⁷Li reaction from 30 ppm BSH or BPA induced an apparent RBE of about 2.2 for the production of dicentrics as compared to exposure to thermal neutrons alone. Since the apparent RBE value is defined as the product of the RBE of a thermal neutron dose alone times a boron localization factor which depends on the concentration of a ¹⁰B-containing compound, this localization factor determines the biological effectiveness of the ¹⁰B(n, α)⁷Li reaction.     

RBEs of Thermal Neutron Capture Therapy and 10B(n,α)7 Li Reaction on Melanoma-Bearing Hamsters

Using Greene's melanoma transplanted into Syrian (golden) hamsters, we determined the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) using ¹⁰B-paraboronophenylalanine (¹⁰B-BPA) in comparison with a 9-MeV electron beam. We also obtained the RBE of the ¹⁰B(n,α)⁷Li reaction by calculation based on summed dose data from TNCT. Throughout this study, the Kyoto University Research Reactor was used as the source for thermal neutrons; the reactor was specially altered to attain a low contamination level both for gamma-rays and fast neutrons. ¹⁰B-BPA was administered 8 hours before thermal neutron irradiation to the hamsters with melanoma. The tumor was then irradiated at 5 MW for 90 minutes. The absorbed dose from this TNCT was calculated by the method of Fairchild and Goodman (Phys. Med. Biol. 1966; 2:15–30). The RBEs of the TNCT and the ¹⁰B(n,α)⁷Li reaction obtained by the tumor growth delay time (TGDT) method were 2.22 and 2.51, respectively, at 10.5 days of TGDT. These RBE values varied with TGDT and the absorbed dose. The RBE value of TNCT had a peak at 7.0 days of TGDT; that of the ¹⁰B(n,α)⁷Li reaction was higher at a low absorbed dose level and lower at a high absorbed dose level.     

Computation of cell survival in heavy ion beams for therapy

A simplified method for the calculation of mammalian cell survival after charged particle irradiation is presented that is based on the track structure model of Scholz and Kraft [1, 2]. Utilizing a modified linear-quadratic relation for the x-ray survival curve, one finds that the model yields linear-quadratic relations also for heavy ion irradiation. If survival is calculated as a function of specific energy, z, in the cell nucleus – thus reducing the stochastic fluctuations of energy deposition – the increase in slope of the survival curve and therefore the coefficient β _z can be estimated with sufficient accuracy from the initial slope, α _z . This permits the tabulation of the coefficients α _z for the particle types and energies of interest, and subsequent fast calculations of survival levels at any point in a mixed particle beam. The complexity of the calculations can thereby be reduced in a wide range of applications, which permits the rapid calculations that are required for treatment planning in heavy ion therapy. The validity of the modified computations is assessed by the comparison with explicit calculations in terms of the original model and with experimental results for track-segment conditions. The model is then used to analyze the influence of beam fragmentation on the biological effect of charged particle beams penetrating to different depths in tissue. In addition, cell-survival rates after neutron irradiation are computed from the slowing-down spectra of secondary charged particles and are compared to experimental observations. Received: 10 August 1996 / Accepted in revised form: 16 December 1996     

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

Aim

In this study, we investigated γH2AX foci as markers of DSBs in normal brain and brain tumor tissue in mouse after BNCT.

Background

Boron 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 methods

We analyzed the number of γH2AX foci by immunohistochemistry 30 min or 24 h after neutron irradiation.

Results

In both normal brain and brain tumor, γH2AX foci induced by ¹⁰B(n,α)⁷Li reaction remained 24 h after neutron beam irradiation. In contrast, γH2AX foci produced by γ-ray irradiation at contaminated dose in BNCT disappeared 24 h after irradiation in these tissues.

Conclusion

DSBs 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 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.     

In vitro and in vivo studies of boron neutron capture therapy: boron uptake/washout and cell death

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on thermal-neutron irradiation of cells enriched with (10)B, which produces α particles and (7)Li ions of short range and high biological effectiveness. The selective uptake of boron by tumor cells is a crucial issue for BNCT, and studies of boron uptake and washout associated with cell survival studies can be of great help in developing clinical applications. In this work, boron uptake and washout were characterized both in vitro for the DHDK12TRb (DHD) rat colon carcinoma cell line and in vivo using rats bearing liver metastases from DHD cells. Despite a remarkable uptake, a large boron release was observed after removal of the boron-enriched medium from in vitro cell cultures. However, analysis of boron washout after rat liver perfusion in vivo did not show a significant boron release, suggesting that organ perfusion does not limit the therapeutic effectiveness of the treatment. The survival of boron-loaded cells exposed to thermal neutrons was also assessed; the results indicated that the removal of extracellular boron does not limit treatment effectiveness if adequate amounts of boron are delivered and if the cells are kept at low temperature. Cell survival was also investigated theoretically using a mechanistic model/Monte Carlo code originally developed for radiation-induced chromosome aberrations and extended here to cell death; good agreement between simulation outcomes and experimental data was obtained.     

Review: Biological Effectiveness of Thermal Neutrons and 10B(n,α)7Li Reaction on Cultured Cells

There are only a few reports on the relative biological effectiveness (RBE) of thermal neutrons and ¹⁰B(n,α)⁷Li reactions either in vitro or in vivo. The data in this paper summarize almost all previously published in vitro data. Because only a few reactors are available for biomedical purposes, it is difficult to make a comparison of data from experiments using the same kind of radiation, and also to make a comparison of data from experiments using the different kinds of radiations. However, it is indispensable for boron neutron capture therapy to make a radiobiological analysis. More intensive study, including repair process and oxygen effect, is necessary for establishing the fundamental basis of the clinical application of boron neutron capture therapy.     

Sublethal and potentially lethal damage repair on thermal neutron capture therapy

Tonicity shock or caffeine postirradiation treatment makes evident fast-type potentially lethal damage (PLD). Caffeine expresses fast-type PLD more efficiently than tonicity shock in X-irradiated B-16 mouse melanoma cells, compared with V79 Chinese hamster cells. The survival curves of thermal neutrons for either V79 or B-16 cells exhibit no shoulder. Neither V79 nor B-16 cells show the sublethal damage (SLD) repair of thermal neutrons. Caffeine-sensitive fast-type PLD repairs exist in X-irradiated B-16 cells, as well as V79 cells. The fast-type PLD repair of B-16 cells exposed to thermal neutrons alone is rather less than that of X-irradiated cells. Furthermore, an extremely low level of fast-type PLD repair of B-16 cells with 10B1-paraboronophenylalanine (BPA) preincubation (20 hours) followed by thermal neutron irradiation indicated that 10B(n,alpha)7Li reaction effectively eradicates actively growing melanoma cells. The plateau-phase B-16 cells are well able to repair the slow-type PLD of X-rays. However, cells can not repair the slow-type PLD induced by thermal neutron irradiation with or without 10B1-BPA preincubation. These results suggest that thermal neutron capture therapy can effectively kill radioresistant melanoma cells in both proliferating and quiescent phases.     

Improvement of the boron neutron capture therapy (BNCT) by the previous administration of the histone deacetylase inhibitor sodium butyrate for the treatment of thyroid carcinoma

We have shown that boron neutron capture therapy (BNCT) could be an alternative for the treatment of poorly differentiated thyroid carcinoma (PDTC). Histone deacetylase inhibitors (HDACI) like sodium butyrate (NaB) cause hyperacetylation of histone proteins and show capacity to increase the gamma irradiation effect. The purpose of these studies was to investigate the use of the NaB as a radiosensitizer of the BNCT for PDTC. Follicular thyroid carcinoma cells (WRO) and rat thyroid epithelial cells (FRTL-5) were incubated with 1 mM NaB and then treated with boronophenylalanine ¹⁰BPA (10 μg ¹⁰B ml^?1) + neutrons, or with 2, 4-bis (α,β-dihydroxyethyl)-deutero-porphyrin IX ¹⁰BOPP (10 μg¹⁰B ml^?1) + neutrons, or with a neutron beam alone. The cells were irradiated in the thermal column facility of the RA-3 reactor (flux = (1.0 ± 0.1) × 10¹⁰ n cm^?2 s^?1). Cell survival decreased as a function of the physical absorbed dose in both cell lines. Moreover, the addition of NaB decreased cell survival (p < 0.05) in WRO cells incubated with both boron compounds. NaB increased the percentage of necrotic and apoptotic cells in both BNCT groups (p < 0.05). An accumulation of cells in G2/M phase at 24 h was observed for all the irradiated groups and the addition of NaB increased this percentage. Biodistribution studies of BPA (350 mg kg^?1 body weight) 24 h after NaB injection were performed. The in vivo studies showed that NaB treatment increases the amount of boron in the tumor at 2-h post-BPA injection (p < 0.01). We conclude that NaB could be used as a radiosensitizer for the treatment of thyroid carcinoma by BNCT.     

Characterization of a UV-resistant strain,UVr-10, established from a human clonal cell line,RSb, with high sensitivity to UV, 4NQO,MNNG and interferon

Characterization was performed of a UV-resistant variant strain, UV^r-10, derived from a human clonal cell line, RSb, with high sensitivity not only to the lethal effect of 254-nm far-ultraviolet (UV) irradiation but also to the effects of 4-nitroquinoline 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and to the cell proliferation inhibition (CPI) effect of human leukocyte interferon (HuIFN-α) preparations.Colony-formation assays confirmed the increased resistance of UV^r-10 cells to both UV and 4NQO, but no increased resistance to MNNG. The marked recovery from the inhibition of the total cellular DNA synthesis of UV^r-10 cells, estimated by [methyl-³H]thymidine ([³H]dThd) uptake into the cellular DNA materials, was seen during 6 h after irradiation or 4NQO treatment even under the conditions without the recovery uptake into those of the parent RSb cells, but not during 6 h after MNNG treatment. Comparative studies on the activity of DNA repair synthesis between UV^r-10 and RSb cells, by measuring the extent of UV-, 4NQO- or MNNG-induced unscheduled DNA synthesis (UDS) and DNA repair replication, revealed an increased activity of UV^r-10 cells to UV and 4NQO but no significant increase of the activity to MNNG. These results suggest that increased DNA repair activities of a UV^r-10 cell line may account for its becoming resistant to the lethal effect of UV and 4NQO.Concerning the CPI effect of HuIFN-α, UV^r-10 cells showed increased resistance. Further, the DNA synthesis activity of UV^r-10 cells was not so inhibited by HuIFN-α exposure as that of RSb cells. However, HuIFN-α-exposed UV^r-10 cells showed more enhanced levels of activity of pppA(2′p5′A)_n synthetase (2–5A synthetase) than the exposed RSb, thus suggesting that HuIFN-α could exert enough intracellular effect even in UV^r-10 cells.The implication of the increased resistance of UV^r-10 cells to the effects of UV, 4NQO and HuIFN-α, but not to those of MNNG, is discussed.     

In vitro studies of DNA damage and repair mechanisms induced by BNCT in a poorly differentiated thyroid carcinoma cell line

Boron neutron capture therapy (BNCT) for aggressive tumors is based on nuclear reaction [¹⁰B (n, α) ⁷Li]. Previously, we demonstrated that BNCT could be applied for the treatment of undifferentiated thyroid carcinoma. The aim of the present study was to describe the DNA damage pattern and the repair pathways that are activated by BNCT in thyroid cells. We analyzed γH2AX foci and the expression of Ku70, Rad51 and Rad54, main effector enzymes of non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, respectively, in thyroid follicular carcinoma cells. The studied groups were: (1) C [no irradiation], (2) gamma [⁶⁰Co source], (3) N [neutron beam alone], (4) BNCT [neutron beam plus 10 µg ¹⁰B/ml of boronphenylalanine (¹⁰BPA)]. The total absorbed dose was always 3 Gy. The results showed that the number of nuclear γH2AX foci was higher in the gamma group than in the N and BNCT groups (30 min–24 h) (p?<?0.001). However, the focus size was significantly larger in BNCT compared to other groups (p?<?0.01). The analysis of repair enzymes showed a significant increase in Rad51 and Rad54 mRNA at 4 and 6 h, respectively; in both N and BNCT groups and the expression of Ku70 did not show significant differences between groups. These findings are consistent with an activation of HRR mechanism in thyroid cells. A melanoma cell line showed different DNA damage pattern and activation of both repair pathways. These results will allow us to evaluate different blocking points, to potentiate the damage induced by BNCT.     

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival.We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP⁺). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.     

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy

closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 10¹² neutrons/cm²).     

Effect of gamma-ray irradiation on Escherichia coli motility

The effects of ionizing radiation on bacteria are generally evaluated from the dose-dependent survival ratio, which is determined by colony-forming ability and mutation rate. The mutagenic damage to cellular DNA induced by radiation has been extensively investigated; however, the effects of irradiation on the cellular machinery in situ remain unclear. In the present work, we irradiated Escherichia coli cells in liquid media with gamma rays from ⁶⁰Co (in doses up to 8 kGy). The swimming speeds of the cells were measured using a microscope. We found that the swimming speed was unaltered in cells irradiated with a lethal dose of gamma rays. However, the fraction of motile cells decreased in a dose-dependent manner. Similar results were observed when protein synthesis was inhibited by treatment with kanamycin. Evaluation of bacterial swimming speed and the motile fraction after irradiation revealed that some E. coli cells without the potential of cell growth and division remained motile for several hours after irradiation.     

The Monte Carlo simulation of the biological effect of the 10B(n, alpha)7Li reaction in cells and tissue and its implication for boron neutron capture therapy

The energy deposition in the nucleus of cells exposed to the 10B(n, alpha)7Li neutron capture reaction has been calculated and compared to the measured biological effect of this reaction. It was found that a considerable distribution of hit sizes to the nucleus occurs. The comparison of hit size frequency with the observed survival indicates that not every hit, independent of its size, can lead to cell death. This implies the existence of a hit size effectiveness function. The analysis shows that the location of boron relative to the radiation-sensitive volume of the cell is of great importance and that average dose values alone are of limited use for predicting the biological effect of this reaction. Boron accumulating in the cell nucleus is much more efficient in cell killing than the same amount of boron uniformly distributed; its presence in one cell, however, has little effect on its neighboring cells in a tissue. When boron is present on the cell surface of a tissue (as presumably delivered by antibodies), its cell-killing effect is greatly reduced compared to that in uniform distribution. However, in this case much of the dose to one cell comes from neutron capture reactions occurring on the surface of its neighbor cells. These data have implications for the choice of boron carries in neutron capture therapy. The mathematical analysis carried out here is similar to that proposed recently for low-level exposure effects of radiation, taking mutation and/or carcinogenesis as biological effects. The results here show that high-level exposure to high-LET particles (resulting in cell killing) should be treated in an analogous manner.     

Marrow-dependent cell function in early stages of infection with Listeria monocytogenes.

Mice were infected with either Listeria monocytogenes (LM) or Yersinia pestis EV 76 stain (YP), which are facultative intracellular and extracellular organisms, respectively. Bacterial growth in spleen was determined at various intervals following challenge, focusing particularly on the critical period prior to the emergence of specific immunity. Natural resistance to LM during the first 2 days was diminished by treatment of adult mice with ⁸⁰Sr or silica particles, but not by treatment with lethal total-body irradiation, cortisol, or cyclophosphamide (CY). Early stages of resistance to YP were unaffected by ⁸⁰Sr, but were reduced by lethal total-body irradiation, silica particles, cortisol, and (CY). Infant mice manifested no resistance comparable to that of adults against LM prior to 19 days of age, whereas resistance against YP was attained by 14 days of age. The data are consistent with the hypothesis that marrow-dependent (M) cells function in host defense against early stages of infection with LM but not with YP.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.