Tumor induction in mice after local irradiation with single doses of either carbon-ion beams or gamma rays

Abstract

Purpose: To determine the dose-dependent relative biological effectiveness (RBE) for tumor prevalence in mice receiving single localized doses to their right leg of either carbon ions (15, 45 or 75 keV/μm) or 137Cs gamma rays.

Methods and materials: A total of 1647 female C3H mice were irradiated to their hind legs with a localized dose of either reference gamma rays or 15, 45 or 75 keV/μm carbon-ion beams. Irradiated mice were evaluated for tumors twice a month during their three-year life span, and the dimensions of any tumors found were measured with a caliper. The tumor induction frequency was calculated by Kaplan-Meier analysis.

Results: The incidence of tumors from 50 Gy of 45 keV/μm carbon ions was marginally higher than those from 50 Gy of gamma rays. However, 60 Gy of 15 keV/μm carbon ions induced significantly fewer tumors than did gamma rays. RBE values of 0.87 + 0.12, 1.29 + 0.08 or 2.06 + 0.39 for lifetime tumorigenesis were calculated for 15, 45 or 75 keV/μm carbon-ion beams, respectively. Fibrosarcoma predominated, with no Linear Energy Transfer (LET)-dependent differences in the tumor histology. Experiments measuring the late effect of leg skin shrinkage suggested that the carcinogenic damage of 15 keV/μm carbon ions would be less than that of gamma rays.

Conclusions: We conclude that patients receiving radiation doses to their normal tissues would face less risk of secondary tumor induction by carbon ions of intermediate LET values compared to equivalent doses of photons.     

Chromosomal Aberrations in Normal Human Cells Induced by the Auger Effect via Ca Atoms

Purpose: To quantify the Auger effect on chromosomal aberrations via Ca atoms in human cells.

Material and Methods: Exponentially growing human normal fibroblasts (GM05389) were irradiated with 4.047 (CaK‐P), 4.026 (CaK‐L) and 4.067 (CaK‐H) keV X‐rays (corresponding to the resonance absorption edge of the Ca K‐shell and slightly below and slightly above the edge, respectively) using synchrotron radiation at the photon factory (PF) of the High Energy Accelerator Organization located in Tsukuba. Chromosomal aberrations induced by the irradiation were analyzed by the premature chromosome condensation (PCC) method using calyculin A. The dependency of the chromosomal aberrations on the incubation time post 2?Gy irradiation was observed for each energy. Irradiation using 200?kVp conventional X‐rays was also examined as a reference to CaK irradiation.

Results: 1. Soon after irradiation with 2?Gy, the enhancement ratios of CaK‐H X‐rays to CaK‐L X‐rays were 1.21, 1.51 and 2.70 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK‐P X‐rays to CaK‐L X‐rays were 1.82, 0.98 and 6.30, for breaks/gaps, isochromatid breaks and exchanges, respectively.

2, After a 6‐hr incubation treatment post 2?Gy irradiation, the enhancement ratios of CaK‐H X‐rays to CaK‐L X‐rays were 1.59, 2.03 and 2.14 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK‐P X‐rays to CaK‐L X‐rays were 1.69, 1.66 and 2.00 for breaks/gaps, isochromatid breaks and exchanges, respectively.

3, Soon after irradiation, the ratios of the efficiencies of CaK‐P X‐rays to those of 200?kVp X‐rays were 1.74, 1.29 and 2.51 for breaks/gaps, isochromatid breaks and exchanges, respectively. And after a 6‐hr incubation treatment, the ratios were 5.50, 1.93 and 1.81 for breaks/gaps, isochromatid breaks and exchanges, respectively.

Conclusions: An effective enhancement of chromosomal aberrations, such as breaks/gaps, isochromatid breaks and exchanges, was caused by Ca K‐shell ionization or excitation. Auger electrons emitted by Ca atoms in irradiated cells appear to have an important role in causing this enhancement. Comparing these efficiencies of chromosomal aberrations with those produced by 200?kVp conventional X‐rays suggests un‐repaired and complicated damage is induced by the X‐rays around the Ca K‐shell resonance absorption edge.     

Influence of the sampling time on chromosomal aberrations at G2 phase in Syrian hamster embryonic cells irradiated with different types of radiation

PURPOSE: To investigate the time-course of chromosomal aberrations following radiations of differing LET. MATERIALS AND METHODS: Syrian hamster embryonic cells were irradiated with nitrogen ions (LET(infinity) = 530 keV/microm) and helium-ions (LET(infinity) = 36 and 77 keV/microm), also 137Cs gamma-rays as a reference radiation. The frequency of chromatid-type aberrations was determined after 0-6 h incubation in a 5% CO2 incubator at 37 degrees C. RESULTS: The amount of chromosomal damage per cell for nitrogen ions detected immediately after irradiation was lower than induced by 137Cs gamma-rays. In contrast, helium ions were more effective than gamma rays in inducing chromatid type damage. The RBE values for the nitrogen-ion beams were 0.45 for gaps, 0.43 for deletions and 0.20 for exchanges. For helium-ion beams, the RBE values for the 36 keV/microm beams and the 77 keV/microm beams were 1.2 and 1.5 for gaps, 1.3 and 2.1 for deletions, and 1.5 and 1.9 for exchanges, respectively. The frequency of cells with chromosomal damage following exposure to gamma-rays and helium-ion beams showed a downward trend with increasing incubation period. In contrast, in the case of nitrogen-ion beams, there was an increase with the incubation period. CONCLUSIONS: The results show that it is possible to underestimate chromosomal damage for different types of radiation by scoring aberrations at a single fixed sampling time.     

Cell cycle arrest and aberration yield in normal human fibroblasts. I. Effects of X‐rays and 195 MeV u−1 C ions

Purpose: To examine the relationship between cell proliferation and the expression of chromosomal damage in normal human skin fibroblasts after X‐ray and particle irradiation.

Materials and methods: Confluent G0/G1 AG1522B cells were exposed to X‐rays or 195?MeV?u^?1 C ions with a linear energy transfer of 16.6?keV?µm^?1 in the dose range 1–4?Gy. Directly after irradiation, cells were reseeded at a low density in medium containing 5‐bromo‐2′‐deoxyuridine. At multiple time points post‐irradiation, the cumulative BrdU‐labelling index, mitotic index and aberration frequency were measured. Based on these data, the total amount of damage induced within the entire cell population was estimated by means of mathematical analysis.

Results: Both types of radiation exposure exert a pronounced effect on the cell cycle progression of fibroblasts. They result in delayed entry of cells into S‐phase and into the first mitosis, and cause a dramatic reduction in mitotic activity. Measurement of chromosomal damage in first‐cycle cells at multiple time points post‐irradiation shows that the frequencies of aberrant cells and aberrations increase with time up to twofold for the lower doses. However, for the higher doses, this effect is less pronounced or even disappears. When the data for the whole cell population are analysed, it becomes evident that only a few damaged fibroblasts can progress to the first mitosis, a response attributable at least in part to a long‐term arrest of injured cells in the initial G0/G1‐phase. As observed in other investigations, the effectiveness of 195?MeV?u^?1 C ions was similar or slightly higher than X‐rays for all endpoints studied leading to a relative biological effectiveness in the range 1.0–1.4.

Conclusions: Cell cycle arrests affect the aberration yield observable in normal human fibroblasts at mitosis. The data obtained for the cell population as a whole reveal that injured cells are rapidly removed from the mitotically active population through a chronic cell cycle arrest, which is consistent with other studies that indicate that this response is a specific strategy of fibroblasts to minimize the fixation and propagation of genetic alterations.     

Difference in the induction,but not in the repair,of X‐ray‐ and nitrogen ion‐induced DNA single‐strand breaks as measured using human cell extracts

Purpose: To compare the repair efficiency of X‐ray (low linear energy transfer [LET]) and nitrogen ion (high LET)‐induced single‐strand breaks (SSB) in a human cell‐free end‐joining system.

Materials and methods: SSB were introduced into a bacterial plasmid, pBR322, by X‐rays (4?MeV photons) and nitrogen ions with an LET=125?keV?µm^?1. Repair efficiency was studied under incubation with the protein extracts from human squamous carcinoma cells, UT‐SCC‐5.

Results: A several fold higher dose of nitrogen ion radiation compared with X‐ray radiation was needed to induce a similar loss of supercoiled plasmid DNA. There was no difference in the repair efficiency of SSB induced by these two types of radiation.

Conclusion: The data indicate that X‐rays at 25?Gy and nitroging ions at 100?Gy radiation doses, under condition of low scavenging capacity (10?mM Tris), induce SSB of similar complexity or, alternatively, differences in SSB complexity do not alter the repair rate.     

Cell cycle arrest and aberration yield in normal human fibroblasts. II: Effects of 11 MeV u−1 C ions and 9.9 MeV u−1 Ni ions

Purpose: To investigate further the relationship between high linear energy transfer (LET) induced cell cycle arrests and the yield of chromosome aberrations observable in normal human fibroblasts at the first post-irradiation mitosis.

Materials and methods: Normal human fibroblasts (AG01522C) were exposed in G0/G1 to either 11 MeV u^?1 C ions (LET = 153.5 keV μm^?1) or 9.9 MeV u^?1 Ni ions (LET = 2455 keV μm^?1), subcultured in medium containing 5-Bromo-2'-deoxyuridine (BrdU) and at multiple time-points post-irradiation the yield of chromosomal damage, the mitotic index and the cumulative BrdU-labelling index were determined. Furthermore, a mathematical approach was used to analyse the entire cell population.

Results: Following high LET exposure normal fibroblasts suffer a transient delay into S-phase and into mitosis as well as a prolonged, probably permanent cell cycle arrest in the initial G0/G1-phase. Cells that reach the first mitosis at early times carried less aberrations than those collected at later times indicating a relationship between cell cycle delay and the number of aberrations. However, with respect to the whole cell population, only a few aberrant fibroblasts are able to progress to the first mitosis. For all endpoints studied the relative biological effectiveness (RBE) of C ions is in the range of 2 – 4, while for Ni ions RBE < 1 is estimated. In contrast, when compared on a per particle basis Ni ions with the higher ionization density were found to be more effective.

Conclusions: Detailed analysis of the data demonstrates that the number of fibroblasts at risk for neoplastic transformation is significantly reduced by a chronic cell cycle arrest in the initial G0/G1-phase and, for the first time, the LET-dependence of this effect has been shown.     

Studies on radiation-induced apoptosis in G0 human lymphocytes

Purpose: To determine the relationships between the frequencies of radiation-induced chromosomal alterations and the extent of apoptosis in G₀ human lymphocytes.

Material and methods: G₀ human peripheral blood lymphocytes (HPBL) were X or γ-irradiated, in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). Directly after irradiation, a part of the lymphocytes were stimulated to grow while the rest were stimulated 48 h after irradiation. These lymphocyte cultures were analysed for induction of chromosomal aberrations. A subset of lymphocytes was kept in G₀ and analysed for cell viability, apoptosis and p53 expression.

Results: The fraction of cells bearing dicentrics was reduced in lymphocytes stimulated to grow 48 h post irradiation as compared to lymphocytes stimulated immediately after irradiation. The decrease in the frequency of dicentrics correlated with the increase in the number of apoptotic cells. The operative apoptotic pathway in irradiated Go lymphocytes was dependent on the expression of p53.

Conclusions: The radiation-induced apoptotic response of G₀ lymphocytes is p53 dependent and increases with the time they are held in G₀. When mitogen was added 48 h after irradiation, cells with dicentrics were either preferentially eliminated or did not enter mitosis. Thus the radiation-induced damage can be underevaluated depending on the time between radiation exposure and the induction of proliferation. These results may have relevance for biodosimetry studies or for evaluations of the efficacy of radiotherapy which are based on the frequencies of chromosomal aberrations.     

Role of DNA-PKcs in the bystander effect after low- or high-LET irradiation

Purpose:?To investigate the role of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the medium-mediated bystander effect for chromosomal aberrations induced by low-linear energy transfer (LET) X-rays and high-LET heavy ions in normal human fibroblast cells.

Materials and methods:?The recipient cells were treated for 12 h with conditioned medium, which was harvested from donor cells at 24 h after exposure to 10 Gy of soft X-rays (5 keV/µm) and ²⁰Ne ions (437 keV/µm), followed by analyses of chromosome aberrations in recipient cells with premature chromosome condensation methods. To examine the role of DNA-PKcs and nitric oxide (NO), cells were treated with its inhibitor LY294002 (LY) and its scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO), respectively.

Results:?Increased frequency of chromosome aberrations in recipient cells treated with conditioned medium from irradiated but not from un-irradiated donor cells was observed which was independent of radiation type. Bystander induction of chromosome aberrations in recipient cells was mitigated when donor cells were treated with LY before irradiation and with c-PTIO after irradiation, and was enhanced when recipient cells were treated with LY before treatment of recipient cells with conditioned medium from irradiated donor cells.

Conclusion:?Irradiated normal human cells secrete NO and other molecules which in turn transmit radiation signals to un-irradiated bystander cells, leading to the induction of bystander chromosome aberrations partially repairable by DNA-PKcs-mediated DNA damage repair machinery, such as non-homologous end-joining repair pathways.     

Biological effectiveness of 12C and 20Ne ions with very high LET

Purpose: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of ¹²C and ²⁰Ne ions.

Materials and methods: Chinese hamster ovary (CHO-K1) cells were exposed to high LET ¹²C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and ²⁰Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up.

Results: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440–830 keV/μm for ¹²C and for 1020–1600 keV/μm for ²⁰Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET.

Conclusions: The RBE decreases with increasing LET in the range between 440 and 1600 keV/μm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.     

Direct and bystander effects in human blood lymphocytes exposed to 241Am alpha particles and the relative biological effectiveness using chromosomal aberration and micronucleus assay

Abstract

Purpose: It is important to understand the significance of alpha (α) radiation-induced bystander effects (RIBE) and its relative biological effectiveness (RBE); this is because the phenomenon is not universal and the mechanism is unclear and because the RBE is widely varying and projected to be very high.

Materials and methods: Isolated lymphocytes from healthy volunteers (n?=?10) were exposed to either low fluence α-particles (²⁴¹Am), γ-rays (⁶⁰Co), or X-rays (225 kVp and 6?MV). Co-culture methodology was employed to investigate bystander effects (BEs). Chromosomal aberrations (CA) and micronucleus (MN) formation were used to study the BE and calculated RBE.

Results: Lymphocytes directly exposed to the types of radiation used showed a dose-dependent increase in the frequency of CA and MN; dose independent increases in the frequency of these chromosomal damages in co-cultured bystander cells, implies that all three types of radiation-induced a BE. The calculated RBE at the level of 5% induced aberrations varied between 9 and 20.

Conclusion: The magnitude of low fluence α-particle induced RIBE is higher than in low LET (linear energy transfer) radiation. The RBE also varies depending upon the endpoints used and adds up to targeted effects. Since the endpoint of CA is considered as an important and early marker of risk prediction, the RIBE and RBE using CA as a marker are relevant for radiation protection purposes.     

X‐irradiated human lymphocytes with unstable aberrations and their preferential elimination by p53/survivin‐dependent apoptosis

Purpose: To investigate whether unstable types of chromosomal aberrations are more effective in priming apoptotic cell death in comparison with stable ones. Also, to highlight the phase of the cell cycle at which apoptosis occurs and the mechanism of its execution.

Materials and methods: G0 human peripheral blood lymphocytes were X‐irradiated in the presence or absence of the repair inhibitor cytosine arabinoside (Ara‐C). After irradiation, the lymphocytes were analysed for induction of dicentrics, translocations, apoptosis, p53 and survivin expression at various recovery times.

Results: A preferential elimination of cells bearing dicentrics with respect to those with balanced translocations was observed. There was a time‐dependent correlation between the decrease in the frequency of dicentrics and the increase in the per cent of apoptotic cells. Most of the apoptotic cells were labelled with bromodeoxyuridine and were mononucleated in cytochalasin B‐treated cells cultures (blocked cytokinesis). However, after continuous colcemid treatment, the apoptotic pathway was not induced. Moreover, in the G2/M‐phase, an increase in p53 and a decrease in survivin occurred that were X‐ray and Ara‐C dose dependent.

Conclusions: The apoptotic process is primed when the dicentric‐bearing human peripheral blood lymphocytes attempt to exit from metaphase. It is possible that unstable aberrations generate changes in the mitotic spindle causing mechanical tension at the kinetochore, activating the mitotic checkpoint and the execution of p53/survivin‐dependent apoptosis.     

Biological characteristics of carbon-ion therapy

Purpose: Radiotherapy using charged and/or high-linear energy transfer (LET) particles has a long history, starting with proton beams up to now carbon-ions. Radiation quality of particle beams is different from conventional photons, and therefore the biological effects of high-LET irradiation have attracted scientific interests of many scientists in basic and clinical fields. A brief history of particle radiotherapy in the past half-century is followed by the reviewed biological effectiveness of high-LET charged particles.

Results: The latter includes 54 papers presenting 506 RBE (relative biological effectiveness) values for carbon ions and a total of 290 RBE values for other ions identified from 48 papers. By setting a selection window of LET up to 100 keV/μm, we fitted a linear regression line to an LET-RBE relation. The resulting slope of the regression line had a dimension of μm/keV, and showed different steepness for different cells/tissues and endpoints as well. The steepest regression was found for chromosome aberration of human malignant melanoma while the shallowest was for apoptosis of rodent cells/tissue. Both tumour and normal tissue showed relatively shallower slopes than colony formation.

Conclusions: In general, there is a large variation of slope values, but the majority (25 out of 29 values) of data was smaller than 0.05 μm/keV.     

Chromosome aberrations and cell inactivation induced in mammalian cells by ultrasoft X‐rays: correlation with the core ionizations in DNA

Purpose: To study the frequency of chromosome aberrations induced by soft X‐rays. To see if the core ionization of DNA atoms is involved in this end‐point as much as it appears to be in cell killing.

Materials and methods: V79 hamster cells were irradiated by synchrotron radiation photons iso‐attenuated in the cell (250, 350, 810?eV). The morphological chromosome aberrations detected in the first post‐irradiation cell division (dicentrics and centric rings) were studied by Giemsa staining.

Results: The chromosome aberrations at 350?eV were, respectively, 2.6±0.8 and 2.1±0.8 times more numerous than at 250 and 810?eV for the same average dose absorbed by the nucleus. These relative effectivenesses are comparable with the ones already measured for cell killing. Moreover, they roughly vary such as the relative numbers of core ionizations (including in the phosphorus L‐shell) produced in DNA and its bound water (water being involved only at 810?eV through the oxygen atoms). In particular, they reproduce the characteristic twofold enhancement at 350?eV, above the carbon K threshold.

Conclusions: Correlations suggest that the core ionization process is likely a common and essential mechanism initiating both chromosome aberration and cell killing end‐points at these photon energies.     

The radiation sensitivity of human chromosomes 2, 8 and 14 in peripheral blood lymphocytes of seven donors

Purpose:?To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable.

Materials and methods:?Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system.

Results:?Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors.

Conclusions:?Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.     

Mutagenic effect of low energy neutrons on human chromosome 11

Purpose: The shape of the dose–effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low‐dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low‐energy neutrons was determined.

Materials and methods: Human–hamster hybrid A_L cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A_L cells were irradiated with neutrons of 0.565, 2.5 or 14.8?MeV and the results were compared with those after 200?kVp X‐rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS).

Results: The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (±2.7) for 0.565?MeV, 10.2 (±1.9) for 2.5?MeV, and 10.2 (±1.6) for 14.8?MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8?MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29?Mbp deletions on average after irradiation with 0.069?Gy of 0.565?MeV neutrons. This scale of deletions is similar to that after 3?Gy 100?kV X‐rays (=34?Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200?kV X‐rays.

Conclusion: Neutrons of low energies (0.565 or 2.5?MeV) produce a linear dose–response for mutation in the tested dose range of 0.015–0.15?Gy. The neutron curve of 14.8?MeV can be approximated by a curvilinear or linear function.     

Chromatin loops are responsible for higher counts of small DNA fragments induced by high-LET radiation,while chromosomal domains do not affect the fragment sizes

Purpose: To apply a polymer model of DNA damage induced by high-LET (linear energy transfer) radiation and determine the influence of chromosomal domains and loops on fragment length distribution.

Materials and methods: The yields of DSB (double-strand breaks) induced by high-LET radiation were calculated using a track structure model along with a polymer model of DNA packed in the cell nucleus. The cell nucleus was constructed to include the chromosomal domains and chromatin loops. The latter were generated by the random walk method.

Results and conclusions: We present data for DSB yields per track per cell, DNA fragment sizes, the radial distribution of DSB with respect to the track center, and the distribution of 0, 1, 2, and more DSB from a single particle. Calculations were carried out for a range of particles including He (40 keV/μm), N (225 keV/μm), and Fe ions (150 keV/μm). Situations relevant to PFGE (pulsed-field gel electrophoresis) and microbeam experiments with direct irradiation of the cell nucleus were simulated to demonstrate the applicability of the model. Data show that chromosomal domains do not have a significant influence on fragment-size distribution, while the presence of DNA loops increases the frequencies of smaller fragments by nearly 30% for fragment sizes in the range from 2 kbp (bp = base pair) to 20 kbp.     

Radiobiological evaluation and correlation with the local effect model (LEM) of carbon ion radiation therapy and temozolomide in glioblastoma cell lines

Purpose:?To investigate the cytotoxic effect of high linear-energy transfer (LET) carbon irradiation on glioblastoma cells lines in combination with temozolomide (TMZ).

Methods and materials:?The cell lines U87-MG expressing wild-type p53 and LN229 expressing both mutant and wild-type p53 were irradiated with monoenergetic carbon ion beams (LET 172 keV/μm) or an extended Bragg peak (LET 103 keV/μm) after treatment with 10 μM or 20 μM TMZ. Cytotoxicity was measured by a clonogenic survival assay, and cell growth as well as cell cycle progression, were examined.

Results:?The p53 mutant was more sensitive to X-ray irradiation than the p53 wild type cell line, which was also expressed in a shorter G2 block. High LET carbon ions show an increased biological effectiveness in both cell lines, which is consistent with the predictive calculations by the Local Effect Model (LEM) introduced by Scholz et al. The cell line LN229 was more sensitive to TMZ treatment than the U87MG cell line expressing wild-type p53 only. The combination of TMZ and irradiation showed an additive effect in both cell lines.

Conclusion:?High LET carbon ion irradiation is significantly more effective for glioblastoma cell lines compared to photon irradiation. An additional treatment with TMZ may offer a great chance especially for several tumor types.     

Low and high LET radiation‐induced apoptosis in M059J and M059K cells

Purpose: To investigate and compare the ability of DNA‐dependent protein kinase (DNA‐PK)‐deficient and ‐proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation.

Materials and methods: A human glioma cell line M059J lacking the catalytic subunit of DNA‐PK (DNA‐PKcs) and its DNA‐PKcs‐proficient counterpart, M059K, were exposed to 1 and 4?Gy of accelerated nitrogen ions (¹⁴N, 140?eV?nm^?1, 8–12?Gy?min^?1) or ⁶⁰Co γ‐rays (0.2?eV?nm^?1, 0.7?Gy?min^?1). The induction of apoptosis was studied up to 144?h post‐irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA‐PKcs and poly(ADP‐ribose) polymerase (PARP) as well as DNA‐PK and caspase‐3 activity were investigated.

Results: Low and high LET radiations (4?Gy) induced a time‐dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144?h post‐irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase‐3 activation.

Conclusions: M059J and M059K cells both display radiation‐induced apoptosis, which occur independently of caspase‐3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.     

Reduction of survival and induction of chromosome aberrations in tobacco irradiated by carbon ions with different linear energy transfers

Purpose : To determine the relationship between linear energy transfer (LET) and the relative biological effectiveness (RBE) for survival reduction and chromosome aberration induction in plants. Materials and methods : Tobacco seeds were exposed to carbon ions having LET ranging from 92 to 260 keV μ m -1. Survival rate was determined at 7 weeks after sowing. Chromosome aberrations were observed when the root length reached about 0.5mm (immediately after radicle emergence), 3 and 10 mm. Results : The RBE for both endpoints increased with increasing LET and showed the highest value at 230 keV μ m -1. The highest RBE was 65.0 for survival reduction and 52.5 for chromosome aberration induction. The types and yield ratio of chromosome aberrations such as fragments and bridges were not affected by radiation type at 0.5mm root length. As the roots elongated from 0.5 to 10 mm, the frequency of aberrant cells gradually decreased. The number of cells with fragments decreased faster than the number of cells with bridges. The decrement of chromosome aberrations appeared to be slower in roots irradiated by carbon ions than in roots irradiated by γ -rays. Conclusions : The results show a close relationship between survival reduction and chromosome aberration induction in plants. The types and yield ratio of initial chromosome aberrations did not differ among γ -rays and carbon ions with different LET.