Radiation-induced gamma-H2AX in mammalian cells irradiated with a synchrotron X-ray microbeam.

In order to study the radiobiological effects from low dose radiation, a cell irradiation system using synchrotron X-ray microbeam has been developed, by which cells can be recognised individually and irradiated one by one with the desired dose of monochromatic X rays. The minimum beam sizes obtained are 2 microm with the focusing optics and 5 microm square with the non-focused beam, and the beam size can be changed easily with a high-precision slit in the case of a non-focused beam. Human fibroblast cells were individually irradiated with this system, and immunostained by gamma-H2AX antibody to visualise the DNA damage. Most of the fluorescent foci were observed in a localised area in cell nuclei, the size of which was almost the same as the beam size.

Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune function in male NC/Nga mice.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunosuppression in humans and animals. However, the effect of TCDD on Th2-type immune responses such as allergic reactions has been unclear. Using NC/Nga mice that developed atopic dermatitis-like skin lesions with marked elevation in plasma of total IgE when bred under conventional conditions, we investigated the effects of a single oral dose of TCDD on immune responses. NC/Nga mice received a single oral dose (0 or 20 microg/kg body weight) of TCDD. On day 7, treatment with TCDD alone decreased the cellularity of thymus. However, treatment with TCDD modified the cellularity of spleens and mesenteric lymph nodes (MLNs) but not of the thymus on day 28. When NC/Nga mice received ip immunization with OVA and alum on the same day as the TCDD treatment (0, 5, or 20 microg/kg body weight), TCDD markedly suppressed the concentrations of Th2-type cytokines (e.g., IL-4 and IL-5) in culture supernatants of spleen cells, whereas IFN-gamma production significantly increased. TCDD exposure reduced anti-OVA and total IgE antibody titers in plasma and did not induce the development of atopic dermatitis-like lesions in the pinnae or dorsal skin of NC/Nga mice. These results suggest that in NC/Nga mice, exposure to TCDD may impair the induction of Th2-type immune responses.

Inactivation of aerobic and hypoxic cells from three different cell lines by accelerated (3)He-, (12)C- and (20)Ne-ion beams.

The LET-RBE spectra for cell killing for cultured mammalian cells exposed to accelerated heavy ions were investigated to design a spread-out Bragg peak beam for cancer therapy at HIMAC, National Institute of Radiological Sciences, Chiba, prior to clinical trials. Cells that originated from a human salivary gland tumor (HSG cells) as well as V79 and T1 cells were exposed to (3)He-, (12)C- and (20)Ne-ion beams with an LET ranging from approximately 20-600 keV/micrometer under both aerobic and hypoxic conditions. Cell survival curves were fitted by equations from the linear-quadratic model and the target model to obtain survival parameters. RBE, OER, alpha and D(0) were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/micrometer, then decreased with a further increase in LET. Clear splits of the LET-RBE or -OER spectra were found among ion species and/or cell lines. At a given LET, the RBE value for (3)He ions was higher than that for the other ions. The position of the maximum RBE shifts to higher LET values for heavier ions. The OER value was 3 for X rays but started to decrease at an LET of around 50 keV/micrometer, passed below 2 at around 100 keV/micrometer, and then reached a minimum above 300 keV/micrometer, but the values remained greater than 1. The OER was significantly lower for (3)He ions than the others.

Cell cycle and LET dependence for radiation-induced mutation: a possible mechanism for reversed dose-rate effect.

A previous study of the mutagenic action of 252Cf radiation in mouse L5178Y cells showed that the mutation frequency was higher when the dose was chronic rather than acute, which was in sharp contrast to the effects reported for gamma-rays (Nakamura and Sawada, 1988). A subsequent study using synchronized cells revealed that the cells at the G2/M stage were uniquely sensitive to mutation induction by 252Cf radiation but not to gamma-rays (Tauchi et al., 1993). A long phase cell population was first subjected to conditioning gamma or 252Cf radiation doses at different dose-rates. The cell cycle distribution of these cells was then observed, and they were then exposed to 252Cf radiation, and the mutation rate was determined. The G2/M fraction increased by 3- to 4-fold when the conditioning doses (2 Gy of gamma or 1 Gy of 252Cf radiation) were delivered chronically over 10 h, but only slightly when the same doses were delivered over a 1 h period or less. Subsequent 252Cf irradiation gave higher mutation frequencies in the cells pre-irradiated with gamma-rays over a protracted period of time than in those exposed with the higher dose-rate gamma-rays. These results suggest that the radiation-induced G2 block could be at least partly (but not totally) responsible for this reverse dose-rate effect (Tauchi et al. 1996). Possible factors which cause the hyper-sensitivity of G2/M cells to mutation induction by neutrons will be discussed.

Phosphatase inhibitors and premature chromosome condensation in human peripheral lymphocytes at different cell-cycle phases.

The cytogenetical reaction of human peripheral lymphocytes to okadaic acid and calyculin A was examined. Calyculin A could induce PCC about 20 times more effectively than okadaic acid. Their mechanisms of PCC induction were judged similar by their dose-dependent manner and chromosome morphology. Contrary to earlier studies suggesting that chemicals could not induce PCC in G1 cells where little cyclin B is present, the present study showed that calyculin A could induce PCC in lymphocytes not only at S and G2/M but also at the second G1 phase after PHA stimulation in vitro. PCC was induced slightly in lymphocytes both at G0 and the first in vitro G1 phase even when the calyculin A concentration increased one hundred fold. It was found that calcium ionophore A23187 increased frequencies of G0-PCC induced by calyculin A, although a further refinement is necessary to obtain a suitable morphology of G0-PCC for cytogenetic studies.

Chemically induced premature chromosome condensation in human fibroblast cell lines: fundamental study for applications to the biodosimetry of local exposure.

The premature chromosome condensation (PCC) of human peripheral lymphocytes treated with inhibitors of protein phosphatase has been demonstrated to be an excellent tool for the estimation of high-dose whole-body exposure. To develop a new biodosimetry for local exposure, the cytogenetical reaction of human fibroblast lines to PCC inducers was examined and compared with that of lymphocytes. The efficiency of the induction by calyculin A was greater than that by okadaic acid in both cell types. Calyculin A induced PCC in 5-Gy-irradiated and unirradiated samples at almost the same frequency in the lymphocytes, whereas the efficacy was considerably lower in irradiated fibroblasts than in unirradiated ones. Calcium ionophore enhanced the induction of PCC in irradiated fibroblasts, although PCC frequencies were still much lower than those in the lymphocytes. The frequency of ring chromosomes observed in 2- and 5-Gy-irradiated fibroblasts was too low to be used as a marker for cytogenetic dosimetry, and that of excess fragments, scored as the observed chromosome number minus 46, might be substituted. The frequency of excess fragments for 2-, 5-, and 10-Gy-irradiated fibroblasts was less than 0.75, about 1 and a few per cell, respectively, although these values changed with the culture period. The prospects and limitations of the application of PCC techniques to fibroblasts are discussed.

Murine cell line SX9 bearing a mutation in the dna-pkcs gene exhibits aberrant V(D)J recombination not only in the coding joint but also in the signal joint.

We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12. 2% as compared with the normal cell line. To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed. SX9 is a novel dna-pkcs-deficient cell line.

Repair of DNA double-strand breaks and cell killing by charged particles.

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing (Kelland et al., 1988; Radford, 1986). We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/micrometer and were even smaller than unity for the LET region greater than 300 keV/micrometer. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/micrometer, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main muse of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.

Irradiation of mixed beam and design of spread-out Bragg peak for heavy-ion radiotherapy.

Data on cellular inactivation resulting from mixed irradiation with charged-particle beams of different linear energy transfer (LET) are needed to design a spread-out Bragg peak (SOBP) for heavy-ion radiotherapy. The present study was designed to study the relationship between the physical (LET) and biological (cell killing) properties by using different monoenergetic beams of 3He, 4He and 12C ions (12 and 18.5 MeV/nucleon) and to attempt to apply the experimental data in the design of the SOBP (3 cm width) with a 135 MeV/nucleon carbon beam. Experimental studies of the physical and biological measurements using sequentially combined irradiation were carried out to establish a close relationship between LET and cell inactivation. The results indicated that the dose-cell survival relationship for the combined high- and low-LET beams could be described by a linear-quadratic (LQ) model, in which new coefficients alpha and beta for the combined irradiation were obtained in terms of dose-averaged alpha and square root of beta for the single irradiation with monoenergetic beams. Based on the relationship obtained, the actual SOBP designed for giving a uniform biological effect at 3 cm depth was tested with the 135 MeV/nucleon carbon beam. The results of measurements of both physical (LET) and biological (90% level of cell killing, etc.) properties clearly demonstrated that the SOBP successfully and satisfactorily retained its high dose localization and uniform depth distribution of the biological effect. Based on the application of these results, more useful refinement and development can be expected for the heavy-ion radiotherapy currently under way at the National Institute of Radiological Sciences, Japan.

The role of DNA repair on cell killing by charged particles.

It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.

Differences in heavy-ion-induced DNA double-strand breaks in a mouse DNA repair-deficient mutant cell line (SL3-147) before and after chromatin proteolysis.

DNA double-strand breaks induced by X- or neon beam-irradiation in a DNA double-strand break-repair-deficient mutant cell line (SL3-147) were examined. The increase in the number of DNA double-strand breaks was dose depend after irradiation with X-rays and neon beams and was enhanced by chromatin-proteolysis treatment before irradiation. These results suggest that the induction of DNA double-strand breaks by ionizing radiation, including heavy-ions, is influenced by the chromatin structure.

Biological effects of active oxygen on an X-ray-sensitive mutant mouse cell line (SL3-147).

The biological effects of active oxygen species were examined in a mutant mouse cell line (SL3-147) that is deficient in the repair of DNA double-strand breaks. This mutant cell line shows different sensitivities to X-rays, hydrogen peroxide, paraquat and menadione when compared to the wild-type cell line (LTA). SL3-147 was more sensitive to X-rays, hydrogen peroxide and paraquat, but was less sensitive to menadione in side by side comparisons to LTA cells. The greater number of DNA double-strand breaks in SL3-147 appears to account for the line's greater sensitivity to X-rays and paraquat. DNA damage other than double-strand breaks or injury to non-DNA targets, however, is responsible for the differences between LTA and SL3-147 in their sensitivities to hydrogen peroxide and menadione.

Interspecific complementation between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation.

Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10.

Reparability of DNA double-strand breaks and radiation sensitivity in five mammalian cell lines.

We examined the relationship between gamma-ray-induced DNA double-strand breaks (dsb) and cell lethality in five mammalian cell lines differing in radiosensitivity; HA-1, HMV-I, L5178Y, M10 and LX830. These cells were derived from Chinese hamster, human melanoma, and mouse lymphoma (parent and its radiosensitive mutants), respectively. HA-1 cells were the most radioresistant and LX830 cells were the most radiosensitive among these five lines. Although the induction of dsb by gamma-rays for HA-1 was significantly different from other curves (p less than 0.05 for HMV-I and p less than 0.01 for L5178Y and M10), those for the other four lines were similar to one another. In addition, the most radioresistant cell line, HA-1, showed the highest dsb induction among five cell lines. Therefore, there is no correlation between radiosensitivity and the induction of dsb in these five lines. On the other hand, residual dsb after repair incubation (non-reparable dsb) do differ from each other. When the relative number of non-reparable dsb was plotted against the radiation dose, the dose-response curves for all the cell lines were concave, and the slopes of curves for M10 and LX830 were steeper than those for other cell lines. These curves are a mirror-image of the survival curves. The results suggest that there is a correlation between the radio-resistance in terms of cell killing and the capacity of cells to repair dsb.

Time-lapse microscopy and DNA double-strand breakage of Chinese hamster cells under conditions promoting or preventing PLD repair after irradiation with 60Co gamma rays.

We have confirmed previous time-lapse microscopic observations (Suzuki 1985) using Chinese hamster hai and V79 cells. The proportion of non-dividing to dividing cells was the same under conditions of potentially lethal damage (PLD) repair and non-PLD repair after irradiation with 60Co gamma-rays. This finding suggested that the radiation-induced damage to cellular DNA was similarly repaired so that cells undergo a first division to the same extent under both sets of conditions. In fact, direct measurement of double-strand breaks (dsb) in DNA from the two cell lines by the neutral elution technique showed no differences either in the initial amount of damage or in the time-course under conditions promoting or preventing PLD repair. These results indicate that PLD repair (i.e. an increase in cell survival) cannot be simply explained by a difference in the repair of dsb, but it can perhaps be explained by assuming that DNA damage is repaired with either fewer or more errors in the presence or absence of PLD repair respectively.

Repair of DNA single- and double-strand breaks in proliferating and quiescent murine tumor cells.

We evaluated the relationship between the repair of DNA single- and double-strand breaks and cellular radiosensitivity in proliferating vs. quiescent cells of the mouse mammary tumor lines 66 and 67 in vitro, using the technique of filter elution at pH 12.2, pH 7.2 and pH 9.6. In these lines, quiescent (Q; unfed plateau-phase) cells are more radiosensitive than are proliferating (P) cells. At doses of 4-6 Gy, both 66 and 67 Q cells repair single-strand breaks (ssb) with kinetics similar to those of P cells. However, repair of ssb was slightly retarded in Q cells at a higher dose (10 Gy) than at the lower doses. In contrast, repair of ssb in P cells was dose-independent, at least for doses up to 10 Gy. The rate of repair of DNA double-strand breaks (dsb), measured at pH 7.2, was dose-independent in P and Q cells of both lines. The repair kinetics were biphasic, with an initial half-time less than 15 min, and the early phase was similar in all cell groups. The half-time for repair in the slow phase ranged from about 2 to greater than 20 h. The fraction of damage repaired by the slow phase was relatively high in all cell groups (40-70 per cent). In line 66, P cells repaired a higher percentage of dsb by 2 h postirradiation than did Q cells. The opposite was observed in line 67: Q cells repaired more dsb in 2 h than did P cells. The survival of 66 St4 cells (Q cultures which have been refed with complete medium and incubated 4 h) was significantly greater than that of 66 Q; nevertheless St4 cells repaired both ssb and dsb at rates similar to those of Q cells. Therefore, survival does not necessarily correlate with the rates of either ssb or dsb repair among these cell lines in different growth states.

DNA-lesion and cell death by alpha-particles and nitrogen ions.

When the natural logarithm of the surviving fraction is plotted against the dose of radiation, curves with shoulders at relatively high survival levels are obtained after gamma-rays. The curves were practically linear in case of HMV-I and HA-1 cells irradiated by charged particle beams. These cells were derived from human malignant melanoma and Chinese hamster cells, respectively. The amount of DNA single strand breaks (ssb) by gamma-rays or nitrogen-ions (LET=530KeV/micrometers) in HMV-I cells increases linearly with increment in dose, when the ssb is detected using the alkaline elution technique. There is no close relationship between the dose-response curve of the ssb and the dose-survival curves after gamma-rays or N-ions. The amount of DNA double strand breaks (dsb) by gamma-rays increases quadratically with increment of dose, in both HMV-I cells and HA-1 cells, when the dsb is detected using the neutral elution technique. The survival fraction for HA-1 cells is slightly higher than that for HMV-I cells, at the same dose, and the amount of dsb for HA-1 cells is considerably greater than that for HMV-I cells. These results suggest that the radiosensitivities to gamma-rays in different cell lines do not correspond to the number of DNA strand breaks. The amount of both non-repairable ssb and dsb also increases quadratically with increment of dose for gamma-rays and almost linearly with increment of dose for N-ions and alpha-particles (LET=36keV/micrometers for HA-1 cells and LET=77keV/micrometers for HMV-I cells). The dose-response curves for non-repairable dsb in case of these radiations seemed to mirror image the dose-survival curves for these radiations, in both cell lines. The number of non-repairable DNA strand breaks in the two cell lines, at the same level of survival was much the same. These results show the close relationship between the induction of non-repairable DNA strand breaks and cell killing.