Y. Furusawa

Nitric oxide-mediated bystander effect induced by heavy-ions in human salivary gland tumour cells

Purpose : To investigate the signal factor and its function in the medium-mediated bystander effect during heavy-ion irradiation of human salivary gland (HSG) neoplastic cells. Materials and methods : Unirradiated recipient HSG cells were co-cultivated with HSG donor cells irradiated with 290 MeV/u carbon beams having different LET values. Cell proliferation and micronucleus (MN) induction in recipient cells with and without treatment of a NO scavenger (PTIO) were measured and the concentration of nitrite in the co-culture medium was detected. As a direct control, the effects of a nitric oxide (NO) generator (sper/NO) on cell proliferation and MN induction were also examined. Results : Increases in cell proliferation and MN induction were found in the recipient HSG cells as a result of co-culturing and cell proliferation was obviously enhanced during a further subculture. In comparison with 13 keV/ μ m, 100 keV/ μ m carbon-ion irradiation was found to be a more efficient inducer of the medium-mediated bystander effect. The treatment of cells by PTIO resulted in elimination of such effects, which supports a role for NO in the medium-mediated bystander effect. As an oxidization product of NO, nitrite was detected in the co-culture medium, and the dose-response for its concentration was similar to that of cell proliferation and MN induction in the recipient cells. When the HSG cells were treated by sper/NO with a concentration of less than 20 μ M, cell proliferation was enhanced, whereas MN increased along with sper/NO concentration. Conclusion : NO participated in the medium-mediated bystander effects on cell proliferation and MN induction, depending on the LET of irradiation.

High- and low-LET induced chromosome damage in human lymphocytes : a time-course of aberrations in metaphase and interphase

Purpose : To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. Materials and methods : Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u -1 iron particles, 400MeV u -1 neon particles or γ-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or γ-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/Hoechst 33258 harlequin staining. Results : There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. Conclusion : The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Induction of radioresistance to accelerated carbon-ion beams in recipient cells by nitric oxide excreted from irradiated donor cells of human glioblastoma.

Purpose : To investigate whether nitric oxide excreted from cells irradiated with accelerated carbon-ion beams modulates cellular radiosensitivity against irradiation in human glioblastoma A-172 and T98G cells. Materials and methods : Western-blot analysis of inducible nitric oxide synthase, hsp72 and p53, the concentration assay of nitrite in medium and cell survival assay after irradiation with accelerated carbon-ion beams were performed. Results : The accumulation of inducible nitric oxide synthase was caused by accelerated carbon-ion beam irradiation of T98G cells but not of A-172 cells. The accumulation of hsp72 and p53 was observed in A-172 cells after exposure to the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase to the medium. The radiosensitivity of A-172 cells was reduced in the conditioned medium of the T98G cells irradiated with accelerated carbonion beams compared with conventional fresh growth medium, and the reduction of radiosensitivity was abolished by the addition of an inducible nitric oxide synthase inhibitor to the conditioned medium. Conclusions : Nitric oxide excreted from the irradiated donor cells with accelerated carbon-ion beams could modulate the radiosensitivity of recipient cells. These findings indicate the importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to accelerated heavy ions.PURPOSE To investigate whether nitric oxide excreted from cells irradiated with accelerated carbon-ion beams modulates cellular radiosensitivity against irradiation in human glioblastoma A-172 and T98G cells. MATERIALS AND METHODS Western-blot analysis of inducible nitric oxide synthase, hsp72 and p53, the concentration assay of nitrite in medium and cell survival assay after irradiation with accelerated carbon-ion beams were performed. RESULTS The accumulation of inducible nitric oxide synthase was caused by accelerated carbon-ion beam irradiation of T98G cells but not of A-172 cells. The accumulation of hsp72 and p53 was observed in A-172 cells after exposure to the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase to the medium. The radiosensitivity of A-172 cells was reduced in the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams compared with conventional fresh growth medium, and the reduction of radiosensitivity was abolished by the addition of an inducible nitric oxide synthase inhibitor to the conditioned medium. CONCLUSIONS Nitric oxide excreted from the irradiated donor cells with accelerated carbon-ion beams could modulate the radiosensitivity of recipient cells. These findings indicate the importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to accelerated heavy ions.

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions

Purpose: This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions – hadrontherapy – with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. Materials and methods: Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 μM PtTC, and then irradiated by fast ions C6+ and He2+, with Linear Energy Transfer (LET) within range 2–70 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). Results: For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. Conclusion: This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts.

Purpose : To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. Material and methods : Exponentially growing human fibroblast cells AG1522 were irradiated with γ-rays, energetic carbon (290 MeV/u), silicon (490 MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. Results : The kinetics of chromatid break rejoining following low-or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10 min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. Conclusions : Compared with γ-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.PURPOSE To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIAL AND METHODS Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290MeV/u), silicon (490MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. RESULTS The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. CONCLUSIONS Compared with gamma-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.

Dose–response of initial G2-chromatid breaks induced in normal human fibroblasts by heavy ions

Purpose : To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of different LET. Material and methods : Exponentially growing human fibroblast cells AG1522 were irradiated with γ-rays, energetic carbon (13 keV/ μ m, 80keV/ μ m), silicon (55 keV/ μ m) and iron (140 keV/ μ m, 185keV/ μ m, 440keV/ μ m) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. Results : The dose-response curves for total chromatid breaks were linear regardless of radiation type. The relative biological effectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55-80 keV/ μ m and decreasing at higher LET. The dose-response curves for isochromatid-type breaks were linear for high-LET radiations, but linear-quadratic for γ-rays and 13 keV/ μ m carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13 keV/ μ m (about 7) and 80 keV/ μ m carbon (about 71), and decreased gradually until 440keV/ μ m iron ions (about 66). Conclusions : High-LET radiations are more effective at inducing isochromatid breaks, while low-LET radiations are more effective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.

p53-dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET

Purpose : To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. Materials and methods : Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/m p53 cells were established by transfection with mutated p53 (m p53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/ neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV w m -1) followed by heating at 44°;C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. Results : It was found that (1) there was no significant difference in cellular sensitivity between SAS/ neo and SAS/m p53 cells to LET radiation of >30 KeV w m -1, although the radiosensitivity of SAS/ neo cells to X-rays was higher (1.2-fold) than that of SAS/m p53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV w m -1 in SAS/ neo cells, although only additive thermal enhancement was observed in SAS/m p53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/ neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/ neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/m p53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. Conclusions : The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.

Apoptosis Induced by High-LET Radiations is not Affected by Cellular p53 Gene Status

To learn more about the biological effects of high-linear energy transfer (LET) radiations, we examined radiation-induced apoptosis in response to high-LET radiations in cells with wild-type, mutated and null p53 gene. Three human lung cancer cell lines were used. These lines had identical genotypes, except for the p53 gene. Cells were exposed to X-rays or high-LET radiations (13 – 200 keV μm−1) using different nuclei ion beams. Cellular radiation sensitivities were determined with the use of colony-forming assays. Apoptosis was detected and quantified using Hoechst 33342 staining with fluorescence microscopy. It was found that (1) there was no significant difference in cellular sensitivity to high-LET radiation (>85 keV μm−1), although the sensitivity of wild-type p53 cells to X-rays was higher than that of mutated p53 or p53-null cells; (2) X-ray-induced apoptosis at higher frequencies in wild-type p53 cells when compared with mutated p53 and p53-null cells; and (3) Fe beams (200 keV μm−1) induced apoptosis in a p53-independent manner. The results indicate that high-LET radiations induces apoptosis in human lung cancer cells in a manner that does not seem to depend on the p53 gene status of the cells.

Irradiation of DNA loaded with platinum containing molecules by fast atomic ions C6+ and Fe26+

Purpose: In order to study the role of the Linear Energy Transfer (LET) of fast atomic ions in platinum-DNA complexes inducing breaks, DNA Plasmids were irradiated by C6+ and Fe26+ ions. Material and methods: DNA Plasmids (pBR322) loaded with different amounts of platinum contained in a terpyridine-platinum molecule (PtTC) were irradiated by C6+ ions and Fe26+ ions. The LET values ranged between 13.4 keV/μm and 550 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added. Results: In all experiments, a significant increase in DNA strand breaks was observed when platinum was present. The yield of breaks induced per Gray decreased when the LET increased. The yield of single and double strand breaks per plasmid per track increased with the LET, indicating that the number of DNA breaks per Gray was related to the number of tracks through the medium. Conclusions: These findings show that more DNA breaks are induced by atomic ions when platinum is present. This effect increases for low LET heavy atoms. As DSB induction may induce cell death, these results could open new perspectives with the association of hadrontherapy and chemotherapy. Thus the therapeutic index might be improved by loading the tumour with platinum salts.

Fast He2+ ion irradiation of DNA loaded with platinum-containing molecules

Purpose: The association of radiotherapy and chemotherapy is an attractive approach to improve the therapeutic index of the treatment of tumors. A lot of work has been devoted to investigate the effects of X-ray, γ-ray and neutron irradiation of DNA or living cells loaded with different chemical compounds containing heavy atoms like platinum. No such studies exist presently when fast atomic ions are chosen as ionizing particles. In the present work, we investigate quantitatively the increase of DNA breaks in complexes of plasmid-DNA loaded with platinum atoms under irradiation by fast atomic He2+ ions. Materials and methods: DNA Plasmids (pBR322) are incubated in solutions containing different concentrations of terpyridine platinum (PtTC). In some preparations, dimethyl sulfoxide (DMSO), a free radical scavenger, has been added in order to investigate the role of the free radicals. The complexes of DNA plasmids loaded with high-Z atoms are irradiated under atmospheric conditions by He2+ ions at an energy of 143 MeV/amu and a linear energy transfert (LET) of 2.24 keV/μm. Analysis of DNA damage – single and double strand breaks – is made by electrophoresis on agarose gels. Results: The results show a significant increase in DNA strand breaks when platinum is present, indicating a radiosensitization by the high Z atoms. The increase in DNA damages is attributed to inner-shell ionization of a platinum atom by secondary electrons emitted along the He2+ tracks followed by an Auger deexcitation, leading, thus, to a local amplification of the radiative effects close to the DNA. The contributions of scavengeable – solvant mediated – indirect effects and non-scavengeable effects (direct ionization) are quantitatively evaluated. Conclusion: Enhancement of DNA breaks in plasmids loaded with heavy atoms like platinum and irradiated by atomic ions are observed. This finding suggests an enhancement of cell death rate will occur under irradiation by atomic ions when the cells contain high-Z atoms located close to DNA due to the increase of the DNA breaks.