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.

Rescue of lethally irradiated mice from hematopoietic death by pre-exposure to 0.5 Gy X rays without recovery from peripheral blood cell depletion and its modification by OK432.

Exposing mice to 0.5 Gy X rays 2 weeks before lethal irradiation has been reported to induce marked radioresistance and to rescue them from hematopoietic death. Here we examined effects of the 0.5-Gy pre-exposure on hematological changes in C57BL mice that were lethally irradiated with 6.5 Gy X rays. Approximately 77% of pre-exposed mice survived 30 days after this irradiation, whereas 80% of mice that did not receive this pre-exposure died by day 20. However, regardless of the pre-exposure, peripheral blood cell counts decreased markedly by day 3 and reached a nadir at day 20. CFU-S in femur and CFU-GM in spleen had started to recover at day 10 and 14, respectively, but recovery of functional peripheral blood cells occurred later. The effect of pre-exposure on survival was altered by OK432, a bioresponse modifier; the effect depended on the timing of its administration. OK432 given 2 days before 0.5 Gy enhanced the protective effect of pre-exposure, resulting in the survival of 97% of the mice. In contrast, injection of OK432 1 day before or 2 days after pre-exposure led to 100% mortality. Thus the survival-promoting effect of 0.5 Gy could be altered by OK432. The OK432-induced changes in the survival of mice could not be attributed solely to hematological changes, as shown by blood cell counts and progenitor cell contents. These results suggest that radioresistance induced by pre-exposure to 0.5 Gy X rays is not stable, but rather varies with the physiological conditions, and can be modulated by factors such as OK432.

Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status.

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53(+/+)) and Trp53 heterozygous (Trp53(+/-)) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53(+/+) embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53(+/+) embryonic mice, while no such effect was found in the Trp53(+/-) embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.

Effects of reoxygenation on repair of potentially lethal radiation damage in cultured MG-63 osteosarcoma cells.

The effects of reoxygenation on repair of potentially lethal radiation damage were investigated using MG-63 human osteosarcoma cells in vitro. When exponentially growing MG-63 cells were cultured under hypoxic conditions for 24 h, cells stopped growing and remained at a low density. The hypoxic cells were then reoxygenated by exposure to air and irradiated with a single dose of 3 Gy X rays. The fraction of the reoxygenated cells surviving after 3 Gy increased by a factor of 20.6 when the colony assay was delayed for 24 h. In control cells which were cultured under aerobic conditions before receiving a single dose of 3 Gy, the surviving fraction increased by a factor of 2.5 when the assay was delayed for 24 h. The difference in the magnitude of the repair observed between reoxygenated and aerobic cells was less prominent in confluent cells plated at high density. The enhanced repair after reoxygenation was due mainly to a decrease in the alpha coefficient when the dose-survival curve was fitted to the linear-quadratic model, whereas the most significant change in the fit of the dose-survival curve for the aerobic cells was a decrease in the beta coefficient. The control aerobic cells accumulated at G2/M phase after irradiation, whereas the reoxygenated cells did not show such an accumulation. When the hypoxic cells were irradiated and then reoxygenated, repair of these cells irradiated under hypoxic conditions was also enhanced. This is the first report to show that reoxygenation could increase cell survival after tumor irradiation.

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.

Adaptive response in embryogenesis: I. Dose and timing of radiation for reduction of prenatal death and congenital malformation during the late period of organogenesis.

An adaptive response was demonstrated during embryogenesis in mice. Whole-body irradiation at a dose of 0-50 cGy was given to condition pregnant ICR mice on day 9 to day 11 of gestation. Then their whole bodies were exposed to a challenging dose of 5 Gy on the next day. The numbers of living fetuses, prenatal deaths and living fetuses with external gross malformations were determined on day 19. A conditioning dose of 30 cGy on day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations induced by a 5-Gy dose on day 12. This indicates the existence of a critical dose and timing for administering a conditioning dose for radioadaptation during the late period of organogenesis in mice. The possible mechanisms involved are discussed.

Adaptive response in embryogenesis: II. Retardation of postnatal development of prenatally irradiated mice.

We previously reported that a priming dose of 0.3 Gy on gestation day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations caused by exposure to 5 Gy X rays on gestation day 12 in ICR mice. In the present study, postnatal development of the live offspring was investigated using a set of developmental and behavioral parameters. The offspring of the mice irradiated with 0.3 Gy generally showed a delay in the appearance of most of the physiological markers, impaired acquisition of neonatal reflexes, and alteration of adult behavior. However, an increase in body weight in the females was observed 4 weeks postnatally. In the offspring primed with 0.3 Gy followed by a challenging dose of 5 Gy prenatally, a high postnatal mortality was found, and all the survivors had various radiation-induced detrimental effects. The results indicated that the priming dose was advantageous to survival itself, but was disadvantageous to the health of survivor. The results also suggested that studying the whole animal can show the extent of the effects of radiation, i.e. quality of life, in a way that cellular or molecular studies cannot.

Radiosensitivity and cell cycle redistribution of cultured human tumour cells during fractionated daily 2-Gy irradiations.

Exponentially growing SQ-5 human lung squamous cell carcinoma cells were irradiated in vitro at 2-Gy per fraction per day continuously for 14 days. The number of total cells continued to increase exponentially till day 5, and reached a plateau level thereafter. The cell cycle distribution changed marginally for the first 5 days, and showed a prominent G2/M accumulation at day 7. Plating efficiencies decreased exponentially with increasing fractionation while the total clonogenic cell number remained constant until day 4. Radiosensitivity at each fraction was stable until day 9, but significantly increased at day 11. A comparison of plating efficiencies between the immediate and 24-h delayed assays revealed that the capacity of cells to spare 2-Gy damage increased with the number of 2-Gy fractions. These results suggest that sublethal damage could accumulate during multifraction daily irradiations, while repair of potentially lethal damage and/or proliferation could rather increase.

Molecular characterization of ionizing radiation-hypersensitive mutant M10 cells.

An ionizing radiation-sensitive mutant derivative of mouse lymphoma L5178Y cell, M10, is defective in rejoining DNA double-strand breaks (DSBs). The complementation test and the results of chromosome transfer suggested that M10 may belong to X-ray cross-complementation (XRCC) group 4. In the present study, sequence analysis of Xrcc4 cDNA in M10 cells disclosed a transversion of A (370) to T, which results in a change of arginine (124) to a termination codon. Interestingly, the mutation occurred in one allele and the transcripts of the Xrcc4 gene were expressed exclusively from the mutant allele. Transfection of M10 cells with the murine Xrcc4 cDNA completely rescued X-ray sensitivity of the mutant cells. M10 is a novel Xrcc4-deficient cell line.

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.

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.

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.

[Radiosensitization of mouse intestinal epithelial cells with BudR].

For the radiosensitization of the repair rich epithelial cells of small intestine, BudR (B.U.) was continuously infused into mice. Its uptake into the DNA, as shown by the substitution rate (S.R.) of thymine to B.U., was dose-dependent in low dose, but above the 4 mg/d/head it did not increased. The radiosensitization effect was assayed by the clonogenecity of the cells, and (1) the repair capacity (recovery factor, RF.) of the cells and (2) the isoeffect dose (I.D) were obtained. From these data, their enhancement ratios (E.R) were estimated. E.R. by R.F. increased remarkably both in low dose and in low S.R., but showed the plateau at about 10 level after 4 mg/d/h of dose or 10% of S.R. Whereas E.R. by I.D. increased linearly in dose or it did quadratically in S.R. From these results, it was discussed that radiosensitization by debromozation of B.U. was seen in low dose, and the direct cytocidal effect of B.U. became dominant with the dose-increase. Also the clinical application of B.U. was discussed in cell kinetical aspect of B.U. labelling as well as the progress of infusion techniques.

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.

Quantitative RT-PCR assay detecting the transcriptional induction of vascular endothelial growth factor under hypoxia.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was used to examine the induction of vascular endothelial growth factor (VEGF) transcript in human osteosarcoma cells, MG-63, under hypoxic culture condition. Using this assay system, the expression of VEGF mRNA was estimated eight-fold higher when cells were cultured under hypoxic condition. Transcription level of hypoxanthine phosphoribosyl transferase (HPRT) mRNA was also examined as an internal control. HPRT mRNA level under hypoxia was reduced to one fourteenth. Secretion of VEGF into the cell culture medium was implied by its stimulating activity on the growth of mouse vascular endothelial cultured cells in vitro.

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.