Equivalency of the quality of sublethal lesions after photons and high-linear energy transfer ion beams.

The quality of the sublethal damage (SLD) after irradiation with high-linear energy transfer (LET) ion beams was investigated with low-LET photons. Chinese hamster V79 cells and human squamous carcinoma SAS cells were first exposed to a priming dose of different ion beams at different LETs at the Heavy Ion Medical Accelerator in the Chiba facility. The cells were kept at room temperature and then exposed to a secondary test dose of X-rays. Based on the repair kinetics study, the surviving fraction of cells quickly increased with the repair time, and reached a plateau in 2-3 h, even when cells had received priming monoenergetic high-LET beams or spread-out Bragg peak beams as well as X-ray irradiation. The shapes of the cell survival curves from the secondary test X-rays, after repair of the damage caused by the high-LET irradiation, were similar to those obtained from cells exposed to primary X-rays only. Complete SLD repairs were observed, even when the LET of the primary ion beams was very high. These results suggest that the SLD caused by high-LET irradiation was repaired well, and likewise, the damage caused by the X-rays. In cells where the ion beam had made a direct hit in the core region in an ion track, lethal damage to the domain was produced, resulting in cell death. On the other hand, in domains that had received a glancing hit in the low-LET penumbra region, the SLD produced was completely repaired.

Gene expression analysis in human malignant melanoma cell lines exposed to carbon beams.

PURPOSE: To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. MATERIALS AND METHODS: We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. RESULTS: Twenty-two genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle - related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including the tumor protein p53 (p53) target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). CONCLUSION: Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle - related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.

Protective effects of melatonin against low- and high-LET irradiation.

To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/microm carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

Repair of skin damage during fractionated irradiation with gamma rays and low-LET carbon ions.

In clinical use of carbon-ion beams, a deep-seated tumor is irradiated with a Spread-Out Bragg peak (SOBP) with a high-LET feature, whereas surface skin is irradiated with an entrance plateau, the LET of which is lower than that of the peak. The repair kinetics of murine skin damage caused by an entrance plateau of carbon ions was compared with that caused by photons using a scheme of daily fractionated doses followed by a top-up dose. Right hind legs received local irradiations with either 20 keV/microm carbon ions or gamma rays. The skin reaction of the irradiated legs was scored every other day up to Day 35 using a scoring scale that consisted of 10 steps, ranging from 0.5 to 5.0. An isoeffect dose to produce a skin reaction score of 3.0 was used to obtain a total dose and a top-up dose for each fractionation. Dependence on a preceding dose and on the time interval of a top-up dose was examined using gamma rays. For fractionated gamma rays, the total dose linearly increased while the top-up dose linearly decreased with an increase in the number of fractions. The magnitude of damage repair depended on the size of dose per fraction, and was larger for 5.2 Gy than 12.5 Gy. The total dose of carbon ions with 5.2 Gy per fraction did not change till 2 fractions, but abruptly increased at the 3rd fraction. Factors such as rapid repopulation, induced repair and cell cycle synchronization are possible explanations for the abrupt increase. As an abrupt increase/decrease of normal tissue damage could be caused by changing the number of fractions in carbon-ion radiotherapy, we conclude that, unlike photon therapy, skin damage should be carefully studied when the number of fractions is changed in new clinical trials.

Arpc1b gene is a candidate prediction marker for choroidal malignant melanomas sensitive to radiotherapy.

PURPOSE: Choroidal malignant melanomas (CMMs) are the most common primary intraocular tumors in adult humans. Although radiotherapy is commonly used to treat the melanomas, the therapeutic effects are unpredictable. The purpose of this study was to search for a gene(s) that can predict the success of radiotherapy for CMMs. METHODS: The cell lines 92-1, OCM-1, and OMM-1 were established from patients with CMM, and radiation sensitivity was determined using the colony-formation assay. RNA was extracted from nonirradiated cells, and gene expression analysis was performed using a microarray containing 10,800 genes. The up- or downregulated genes were verified by real-time PCR using other cancerous cell lines in which radiation sensitivity had been documented. RESULTS: Analysis of radiation survival curves showed that cell line 92-1 was radiation sensitive and OCM-1 and OMM-1 lines were radiation resistant. The results of microarray analyses showed that 34 genes were differentially expressed in the OCM-1 and OMM-1 cell lines compared with the 92-1 cell line. The validity of the expression level of 13 of the 34 genes that were identified by microarray was confirmed by PCR. From the analysis of the different radio-sensitivity cancer cell lines, the Arpc1b gene was selected as a prediction marker gene for sensitivity of CMM to radiotherapy. CONCLUSIONS: Gene expression analysis of CMM cell lines can be used to search for radiation sensitivity prediction markers. Comprehensive gene expression profiles of radiation-sensitive and/or resistant cell lines may provide new insights into the mechanisms of resistance or sensitivity to radiation therapy.

Enhanced efficiency in cell killing at the penetration depths around the Bragg peak of a radioactive 9C-ion beam.

PURPOSE: To evaluate the potential importance of radioactive 9C-ion beam in cancer radiotherapy. METHODS AND MATERIALS: Human salivary gland (HSG) cells were exposed to a double-radiation-source 9C beam at different depths around the Bragg peak. Cell survival fraction was determined by standard clonogenic assay. For comparison, the same experiment was conducted for a therapeutic 12C beam. To determine relative biologic effectiveness (RBE) values, HSG cells were also irradiated with 60Co gamma-rays of fractionation scheme as the reference. RESULTS: The 9C beam was more efficient in cell killing at the depths around its Bragg peak than was the 12C beam, which corresponded to the 9C-ion stopping region and where delayed low-energy particles were emitted. The RBE value at 50% survival level for the 9C beam varied from 1.38 to 4.23. Compared with the 12C beam, the RBE values for the 9C beam were always higher; an increase in RBE by a factor of up to 1.87 has been observed at the depths distal to the Bragg peak. CONCLUSION: The potential advantage of radioactive 9C-ion beam in cancer therapy has been revealed at low dose rate in comparison with a therapeutic 12C beam. This observation, however, remains to be investigated at therapeutic dose rates in the future.

Biological gain of carbon-ion radiotherapy for the early response of tumor growth delay and against early response of skin reaction in mice.

The biological effectiveness of carbon ions relative to gamma rays (RBE) was compared between the tumor growth delay and an early skin reaction of syngeneic mice. The RBE was larger for a tumor than skin when irradiated with large doses of high-LET (linear energy transfer) carbon ions. The intra-track damage (a term of a linear quadratic model) of a tumor and skin increased equally with an increase of the LET, while the inter-track damage (beta term) of skin alone increased with the LET. These data provide evidence that high-LET radiotherapy could achieve therapeutic gain by minimizing the difference in response to fractionated irradiation between the tumor and normal tissue.

Radiosensitization by hyperthermia in the chicken B-lymphocyte cell line DT40 and its derivatives lacking nonhomologous end joining and/or homologous recombination pathways of DNA double-strand break repair.

Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70(-/-), DNA-PKcs(-/-/-), RAD54(-/-) and KU70(-/-)/RAD54(-/-). Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70(-/-)/RAD54(-/-), which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70(-/-) and DNA-PKcs(-/-/-), consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G(1) and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G(2), capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G(1) and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.

High-LET radiation enhanced apoptosis but not necrosis regardless of p53 status.

PURPOSE: We analyzed the death pattern of human lung cancer cells harboring different p53 statuses after irradiation with different levels of linear energy transfer (LET). METHODS AND MATERIALS: We used three kinds of human lung cancer cell lines with identical genotypes, except for the p53 gene. These cells were exposed to X-rays or accelerated carbon-ion beams. The cellular sensitivities were determined by a colony-forming assay. The detection and quantification of cell death (apoptosis and necrosis) were evaluated and compared by acridine orange/ethidium bromide double staining for fluorescence microscopy. RESULTS: We found that (1) there was no significant difference in cellular sensitivity to LET radiation >70 KeV/microm, although wild-type p53 cell sensitivity to X-rays was higher than that of mutated p53 or p53-null cells; (2) low-LET radiation effectively induced apoptosis in wild-type p53 cells as compared with mutated p53 and p53-null cells; and (3) high-LET radiation induced p53-independent apoptosis. CONCLUSIONS: Our findings suggest that high-LET radiotherapy is expected to be a valid application for patients carrying mutated p53 cancer cells. We proposed that the elucidation of the p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.

Prompt tumor formation and maintenance of constitutive NF-kappaB activity of multiple myeloma cells in NOD/SCID/gammacnull mice.

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM-1 and U-266 in an in vivo cell proliferation model using NOD/SCID/gammacnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post-auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM-1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human lambda-chain in various organs of all NOG mice, while the U-266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human lambda Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL-6R and gp130. Tumor cells sustained a strong NF-kappaB activity in vivo and induced NF-kappaB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.

Bystander effect in lymphoma cells vicinal to irradiated neoplastic epithelial cells: nitric oxide is involved.

Evidence has been accumulated for attached cells demonstrating that nonirradiated cells can have a response to the ionization events delivered to their neighbors. In the present study, we first investigated the bystander responses between suspension and neoplastic cells by coculturing L5178Y (LY) cells with human salivary gland (HSG) cells that had been irradiated with either 290 MeV/u carbon ions or X-rays. After this coculture, the survival of nonirradiated recipient LY cells showed dichotomous responses to the irradiation dose delivered to HSG cells. Apoptosis and necrosis were also produced in a 48 h subculture of the recipient LY cells, and their yield increased, but then had a tendency to decrease when the irradiation dose increased. Treatment of cells with PTIO, a nitric oxide specific scavenger, diminished apoptosis and necrosis of the recipient LY cells to the control level. As an oxidization product of NO, nitrite was detected in the coculture medium and its time course corresponded well to the decrease of the viability of irradiated HSG cells. Moreover, the relationship of the survival and the apoptotic and necrotic production of the recipient LY cells to the nitrite concentration followed a linear-quadratic model. The present findings of NO being involved in the radiation-induced bystander effect may have significance in terms of radiotherapy.

Action spectra of apoptosis induction and reproductive cell death in L5178Y cells in the UV-B region.

We investigated the action spectra for the induction of apoptosis and reproductive cell death in mouse lymphoma L5178Y cells using a high-performance spectroirradiator, the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki. L5178Y cells were exposed to monochromatic light at different wavelengths in the UV-B and UV-A regions. The frequencies of apoptosis induction and reproductive cell death were determined by counting cells with chromatin condensation and by a semi-solidified agarose colony formation assay, respectively. The measured action spectra for the two end-points were similar. The sensitivity decreased steeply with an increase of wavelength in the UV-B region, but showed no further decrement in the UV-A region. The action spectra were slightly steeper than that for the minimum erythematic dose (MED), and were similar to the light-absorption spectrum of DNA in the UV-B region. On the other hand, in the UV-A region, the spectra for both endpoints were close to the MED, but not to DNA absorption spectra. The difference between the measured spectra and that for MED may have been caused by the absorption of the light by the skin. Differences in the time course and morphological difference of apoptosis were found between the UV-B and UV-A region. These results suggest that although DNA damage induced by UV-B light can trigger apoptosis, or lead to reproductive cell death, other damage (membrane, protein and so on) may trigger those effects in the UV-A region.

Microbeams of heavy charged particles.

We have established a single cell irradiation system, which allows selected cells to be individually hit with defined number of heavy charged particles, using a collimated heavy-ion microbeam apparatus at JAERI-Takasaki. This system has been developed to study radiobiological processes in hit cells and bystander cells exposed to low dose and low dose-rate high-LET radiations, in ways that cannot be achieved using conventional broad-field exposures. Individual cultured cells grown in special dishes were irradiated in the atmosphere with a single or defined numbers of 18.3 MeV/amu 12C, 13.0 MeV/amu 20Ne, and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells were performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. The actual number of particle tracks that pass through cell nuclei was detected with prompt etching of the bottom of the cell dish made of ion track detector TNF-1 (modified CR-39), with alkaline-ethanol solution at 37 degrees C for 15-30 minutes. Using this system, separately inoculated Chinese hamster ovary cells, confluent normal human fibroblasts, and single plant cells (tobacco protoplasts) have been irradiated. These are the first studies in which single-ion direct hit effect and the bystander effect have been investigated using a high-LET heavy particle microbeam.

Radioprotective activities of beer administration for radiation-induced acute toxicity in mice.

BACKGROUND AND PURPOSE: We previously found that drinking beer reduces chromosome aberrations in blood lymphocytes that were collected and irradiated in vitro. In this study, we investigated the radioprotective activities of beer-administration for bone marrow and intestine in mice. METHODS: C3H/He female mice received an oral administration of beer, ethanol or saline at a dose of 1 ml/mouse 30 min before whole body irradiation with 137Cs gamma rays or LET 50 keV/microm carbon ions. Radioprotective activities were estimated using a LD(50/30) (The dose required to kill 50% of the mice within 30 days) and a microcolony technique for intestine. RESULTS: The LD(50/30) for the beer-administered mice was significantly increased in comparison with saline administered mice. The LD(50/30) of gamma-ray was 7.8 Gy (p < 0.05), 7.6 Gy and 7.3 Gy for beer-, ethanol- and saline-administered group, respectively. The LD(50/30) of carbon ions was 6.6 Gy (p < 0.05), 6.2 Gy and 5.9 Gy for the beer-, ethanol- and saline-administered groups, respectively. The crypt survivals that were semi-logarithmically plotted against dose were well fitted to a linear regression line. The dose reduction factor (DRF) (D10) of beer- and ethanol-administered mice for gamma rays was 1.09 and 1.08, respectively. The DRF (D10) of beer- and ethanol-administered mice for carbon ions was 1.08 and 1.07, respectively. CONCLUSIONS: The radioprotection by beer-administration is due to not only OH radical-scavenge action by the ethanol contained in beer.

Bystander effect on cell growth stimulation in neoplastic HSGc cells induced by heavy-ion irradiation.

The bystander effect on unirradiated neoplastic human salivary gland (HSGc) cells was investigated by co-culturing them with HSGc cells that had been irradiated with 290 MeV/u carbon beams of different linear energy transfer (LET) values. It was found that the plating efficiency and proliferation of the unirradiated recipient cells were increased and that these increases were related to the LET as well as the radiation dose. Exposure of HSGc cells to higher LET and higher dose was much more effective in enhancing the plating efficiency and proliferation of the unirradiated cells than exposure to lower LET and lower dose. However, when PTIO, a nitric oxide (NO)-specific scavenger, was present in the co-culture medium, the cell growth capacity of the unirradiated recipients was reduced to control level, indicating that NO is involved in the bystander response. As an oxidation product of NO, nitrite was detected in the co-culture medium and its concentration depended on the LET and dose of irradiation. Using a NO-generator sper/NO, it was verified that NO at low concentrations indeed enhanced cell proliferation. Accordingly, NO plays an important role in medium-mediated bystander effects.

Role of gap junctional intercellular communication in radiation-induced bystander effects in human fibroblasts.

Involvement of gap junctional intercellular communication (GJIC) in bystander responses of confluent human fibroblasts irradiated with a carbon-ion beam was investigated. It was found that the lower the radiation dose, the higher the yield of radiation-induced micronuclei per nuclear traversal, suggesting the existence of bystander effects. This low-dose sensitivity was increased when GJIC was enhanced by treating cells with 8-Br-cAMP, but it was partly reduced by treating cells with DMSO, an effective scavenger of reactive oxygen species (ROS). Moreover, no low-dose sensitivity was observed when cells were treated with 100 micro M lindane, an inhibitor of GJIC. The survival of irradiated cells was increased by DMSO but was not influenced significantly by cAMP or lindane. On the other hand, G(1)-phase arrest was detected in the irradiated cells, and it was enhanced by cAMP. In contrast, this arrest was reduced or almost eliminated by DMSO or lindane, respectively, even when cells were irradiated with such a high dose that each cell received five nuclear traversals on average. Thus the bystander responses occurred after both low-dose and relatively high-dose irradiation. Our results indicated that both GJIC and ROS contributed to the radiation-induced bystander effect, but gap junctional channels might play an essential role by modulating the release of radiation-induced signaling factors.

Sper/NO-induced reversible proliferation inhibition and cycle arrests associated with a micronucleus induction in HSG cells.

Nitric oxide (NO) is an important messenger molecule with multiple biological activities. In the present study, sper/NO, a NO generator, showed a biphasic effect on the proliferation of human salivary gland neoplastic (HSG) cells. Sper/NO of less than 20 micro M stimulated cells to depart from the G2/M phase and so enhanced cell division and cell proliferation. But sper/NO at higher concentrations restrained cell proliferation and blocked cell-cycle progression. Cells were mainly arrested in the G2/M phase and S phase when they were treated with 100-200 and 300-500 micro M sper/NO, respectively. A special S-phase peak was detected in a histogram of the cell-phase distribution of sper/NO-treated HSG. When the concentration of sper/NO increased, the S-phase peak shifted from early the G2/M-phase to later the G1-S-phase boundary. Sper/NO-induced cell-cycle arrests were reversible when the cells were released from NO stress for 48h and hence cell proliferation was recovered. In addition, micronucleus, but no apoptosis, was produced in the sper/NO-treated cells, and its yield tended to a saturation value with increasing concentrations of sper/NO. The sper/NO-induced effects were effectively eliminated or reduced by treating cells with PTIO, a NO-specific scavenger, indicating that NO is the main source of these effects.

Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center.

PURPOSE: To assess the biologic effects of proton and carbon ion beams before clinical use. METHODS AND MATERIALS: Cultured cells from human salivary gland cancer (HSG cells) were irradiated at 5 points along a 190 MeV per nucleon proton and a 320 MeV per nucleon carbon ion beam, with Bragg peaks modulated to 6 cm widths. A linac 4 MV X-ray was used as a reference. Relative biologic effectiveness (RBE) values at each point were calculated from survival curves. Cells were also irradiated in a cell-stack phantom to identify that localized cell deaths were observed at predefined depth. Total body irradiation of C3H/He mice was performed, and the number of regenerating crypts per jejunal section was compared to calculate intestinal RBE values. For carbon ion and referential 4 MV X-ray beams, mouse right legs were irradiated by four-fractional treatment and followed up for skin reaction scoring. RESULTS: RBE values calculated from cell survival curves at the dose that would reduce cell survival to 10% (D10) ranged from 1.01 to 1.05 for protons and from 1.23 to 2.56 for carbon ions. The cell-stack phantom irradiation revealed localized cell deaths at predefined depth. The intestinal RBE values ranged from 1.01 to 1.08 for protons and from 1.15 to 1.88 for carbon ions. The skin RBE value was 2.16 at C320/6 cm spread-out Bragg peak (SOBP) center. CONCLUSION: The radiobiologic measurements of proton and carbon ion beams at Hyogo Ion Beam Medical Center are consistent with previous reports using proton beams in clinical settings and carbon ion beams with similar linear energy transfer (LET) values.

[Analysis of death pattern in cancer cells by using different kinds of LET radiation].

We investigated the death pattern of cancer cells by using different kinds of linear energy transfer (LET) radiation. We used two human squamous cell carcinoma cell lines with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with the mp53 gene to SAS cells having functional p53 (wtp53). As the control, a neovector was transfected to the SAS cells (SAS/neo cells). Both types of cells were exposed to X-rays (1.5 KeV/micron) or accelerated C-beams (30-100 KeV/micron). The frequency of cell death (apoptosis and necrosis) was measured by acridine orange/ethidium bromide(AO/EB) double staining for fluorescence microscopy. We found that (1) low-LET radiation induced apoptosis only in SAS/neo cells; (2) high-LET radiation at an iso-survival dose induced apoptosis not but necrosis in SAS/neo cells at a higher frequency; (3) high-LET radiation induced p53-independent apoptosis even in SAS/mp53 cells. Our findings suggest that high-LET radiotherapy is expected to (1) have validity in its application to patients carrying mutated p53 cancer cells and (2) reduce injury to adjacent normal tissue for high-frequency-induced apoptosis without inflammatory response. We propose that elucidation of p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.

Effect of a hypoxic cell sensitizer doranidazole on the radiation-induced apoptosis of mouse L5178Y lymphoma cells.

We investigated the sensitizing effect of the 2-nitroimidazole analogue doranidazole, a new hypoxic radiosensitizer, on radiation-induced apoptosis in L5178Y cells. Apoptosis was assessed by checking DNA ladder formation, the presence of sub-G1 peaks in flow cytometry, and chromatin condensation. A radiosensitizing effect of doranidazole was also confirmed by a soft-agar colony assay of surviving cells. In the assay of DNA ladder formation, DNA fragmentation was observed following irradiation under an aerobic or hypoxic condition with or without doranidazole. The proportions of the cells at the sub-G1 peak in a flow cytometric measurement was not very different among the irradiations at 5 Gy under the aerobic condition, 15 Gy under hypoxia, and 10 Gy with 1 mM doranidazole under hypoxia. The fraction of cells with chromatin condensation was found to be significantly increased with doranidazole up to 3 mM when applied under hypoxic irradiation, but did not increase even at 10 mM. The sensitizer enhancement ratio was estimated to be about 1.7 with a concentration of 1 mM. This enhancement ratio was not different from that observed by assaying cell survivals. On the other hand, doranidazole showed no radiosensitizing effect under aerobic conditions with 1 mM. In conclusion, the radiation-induced apoptosis of L5178Y cells was enhanced by doranidazole under hypoxia.