R. Cherubini

RBE-LET relationships for cell inactivation and mutation induced by low energy protons in V79 cells: further results at the LNL facility.

PURPOSE RBE-LET relationships for cell inactivation and hprt mutation in V79 cells have been studied with mono-energetic low-energy proton beams at the radiobiological facility of the INFN-Laboratori Nazionali di Legnaro (LNL), Padova, Italy. MATERIALS AND METHODS V79 cells were irradiated in mono-layer on mylar coated stainless steel petri dishes, in air. Inactivation data were obtained at 7.7, 34.6 and 37.8 keV/microm and hprt mutation was studied at 7 7 and 37.8 keV/microm. Additional data were also collected for both the end points with the proton LET already considered in our previous publications, namely 11.0, 20.0 and 30.5 keV/microm. RESULTS A maximum in the RBE-LET relationship for cell inactivation was found at around 31 keV/microm, while the RBE for mutation induction increased continuously with LET. CONCLUSIONS The proton RBE-LET relationship for cell inactivation is shifted to lower LET values compared with that for heavier ions. For mutation induction, protons of LET equal to 7.7keV/microm gave an RBE value comparable with that obtained by helium ions of about 20 keV/microm. Mutagenicity and lethality caused by protons at low doses in the LET range 7.7-31 keV/microm were proportional, while the data at 37.8 keV/microm suggest that this may not hold at higher LET values.

RBE-LET Relationship for the Survival of V79 Cells Irradiated with Low Energy Protons

The survival of V79 Chinese hamster cells irradiated with proton beams with energies of 0.73, 0.84, 1.16, 1.70 and 3.36 MeV, corresponding to LET values, evaluated at the cell midplane, of 34.5, 30.4, 23.9, 17.8 and 10.6 keV/micron respectively, have been studied in the dose range 0.5-6.0 Gy. As a reference, the survival curve obtained with 200 kV X-rays was used. The initial shoulder, typical of survival curves obtained with sparsely ionizing radiation, decreases as the LET increases and completely disappears at 23.9 keV/micron. This value corresponds to the maximum of the RBE, expressed as the initial slope ratio. In the energy range we have considered, the RBEs for protons are higher than those reported for other ions of comparable LET and the RBE-LET relationship results shifted to lower LET values. Our data seem to indicate that the RBE-LET curve depends on the type of radiation and this could imply that LET is not a good reference for the dose-effectiveness relationship.

Inactivation and Mutation Induction in V79 Cells by Low Energy Protons: Re-evaluation of the Results at the LNL Facility

During the upgrading of the radiobiological facility at the Laboratori Nazionali di Legnaro (LNL) we found that uncorrected values of the proton energy were used in the past. This circumstance prompted us to perform the re-evaluation of the physical parameters for all the proton beams used in our previous radiobiological investigations (Belli et al. 1987) and, subsequently, the re-evaluation of all our previous dose-response curves for inactivation and mutation induction (Belli et al. 1989, 1991). This re-evaluation leads to significant changes in the dose-response curves and in the RBE-LET relationships only at the two lowest energies (highest LET) used. These two points are not reliable for the identification of a peak in RBE-LET relationship for cell inactivation. In spite of that, the extent of the changes is not such as to modify the general conclusion previously drawn, pointing out that there is a LET range where protons are more effective than alpha-particles.

Inactivation of human normal and tumour cells irradiated with low energy protons

Purpose : To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation. Materials and methods : Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with γ-rays and proton beams with linear energy transfer (LET) from 7 to 33keV/ μ m. Clonogenic survival was assessed. Results : Survival curves are reported for each cell line following irradiation with γ-rays and with various proton LETs. The surviving fraction after 2 Gy of γ-rays was 0.72 for SQ20B cells, and 0.28–0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of γ-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with γ-radiation down to 0.37 with 30keV/ μ m protons. The relative biological effectiveness (RBE(2Gy γ) ) with the 30 keV/ μ m beam, evaluated as the ratio of 2Gy to the proton dose producing the same inactivation level as that given by 2 Gy of γ-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively. Conclusions : RBE for inactivation with high-LET protons increased with the cellular radioresistance to γ-rays. The cell line with the greatest resistance to γ-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20–125keV/ μ m on human tumour cell lines.PURPOSE To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation. MATERIALS AND METHODS Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with y-rays and proton beams with linear energy transfer (LET) from 7 to 33 keV/microm. Clonogenic survival was assessed. RESULTS Survival curves are reported for each cell line following irradiation with gamma-rays and with various proton LETs. The surviving fraction after 2 Gy of gamma-rays was 0.72 for SQ20B cells, and 0.28-0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of gamma-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with gamma-radiation down to 0.37 with 30 keV/microm protons. The relative biological effectiveness (RBE(2 Gy gamma)) with the 30 keV/microm beam, evaluated as the ratio of 2 Gy to the proton dose producing the same inactivation level as that given by 2 Gy of gamma-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively. CONCLUSIONS RBE for inactivation with high-LET protons increased with the cellular radioresistance to gamma-rays. The cell line with the greatest resistance to gamma-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20-125 keV/microm on human tumour cell lines.

DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study.

Purpose : To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). Materials and methods : V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). Results : The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. Conclusions : The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.PURPOSE To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). MATERIALS AND METHODS V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). RESULTS The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2 h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. CONCLUSIONS The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.

DNA DSB induced in human cells by charged particles and gamma rays: Experimental results and theoretical approaches

Purpose:To quantify the role played by radiation track structure and background fragments in modulating DNA fragmentation in human cells exposed to γ-rays and light ions. Materials and methods: Human fibroblasts were exposed in vitro to different doses (in the range from 40 – 200 Gy) of 60Co γ-rays and 0.84 MeV protons (Linear Energy Transfer, LET, in tissue 28.5 keV/μm). The resulting DNA fragments were scored under two electrophoretic conditions, in order to optimize separation in the size ranges 0.023 – 1.0 Mbp and 1.0 – 5.7 Mbp. In parallel, DNA fragmentation was simulated both with a phenomenological approach based on the “generalized broken-stick” model, and with a mechanistic approach based on the PARTRAC (acronym of PARticle TRACk) Monte Carlo code (1.32 MeV photons were used for the simulation of 60Co γ-rays). Results: For both γ-rays and protons, the experimental dose response in the range 0.023 – 5.7 Mbp could be approximated as a straight line, the slope of which provided a yield of (5.3 ± 0.4) • 10−9 Gy−1 bp−1 for γ-rays and (7.1 ± 0.6) • 10−9 Gy−1 bp−1 for protons, leading to a Relative Biological Effectiveness (RBE) of 1.3 ± 0.2. From both theoretical analyses it appeared that, while γ-ray data were consistent with double-strand breaks (DSB) random induction, protons at low doses showed significant deviation from randomness, implying enhanced production of small fragments in the low molecular weight part of the experimental range. The theoretical analysis of fragment production was then extended to ranges where data were not available, i.e. to fragments larger than 5.7 Mbp and smaller than 23 kbp. The main outcome was that small fragments (<23 kbp) are produced almost exclusively via non-random processes, since their number is considerably higher than that produced by a random insertion of DSB. Furthermore, for protons the number of these small fragments is a significant fraction (about 20%) of the total number of fragments; these fragments remain undetected in these experiments. Calculations for 3.3 MeV alpha particle irradiation (for which no experimental data were available) were performed to further investigate the role of fragments smaller than 23 kbp; in this case, besides the non-random character of their production, their number resulted to be at least as much as half of the total number of fragments. Conclusion: Comparison between experimental data and two different theoretical approaches provided further support to the hypothesis of an important role of track structure in modulating DNA damage. According to the theoretical approaches, non-randomness of fragment production was found for proton irradiation for the smaller fragments in the experimental size range and, in a significantly larger extent, for fragments of size less than 23 kbp, both for protons and alpha particles.

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. I. Experimental results.

Purpose : To compare the results on DNA fragmentation induced in Chinese hamster V79 cells by various doses of γ-rays and low-energy protons and helium-4 ions. Materials and methods : V79 cells were irradiated as monolayers with monoenergetic protons and helium-4 ions; γ-rays were used as the reference radiation. DNA double-strand breaks were evaluated by calibrated pulsed-field gel electrophoresis using conditions covering the range 5.7 Mbp-23.1 kbp. Results : The fragment-counting method gave double-strand breaks yields and the relative biological effectiveness higher than those obtained by the fraction of activity released method. The frequency distribution of fragments showed that protons and helium ions induced more fragments below the Mbp region than did γ-rays at the same dose. The distributions for both the irradiated and non-irradiated samples clearly appeared to be non-random. Conclusion : Differences were observed in the yield and spatial correlation, at a molecular size scale characteristic of loop dimensions, of the double-strand breaks induced by γ-rays and by light ions. These effects may have a role in the observed different cell response to these radiations.

Micronuclei, CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality

PURPOSE To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. MATERIALS AND METHODS Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/microm and 27.6 keV/microm protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. RESULTS MN yield and the frequency of BN cells carrying multiple MN (> or =2) were significantly higher after exposure to 27.6 keV/microm protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/microm protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6 keV/microm protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3 Gy). CONCLUSIONS Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.Purpose : To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. Materials and methods : Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/mum and 27.6 keV/mum protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. Results : MN yield and the frequency of BN cells carrying multiple MN (2) were significantly higher after exposure to 27.6 keV/mum protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/mum protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6keV/mum protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3Gy). Conclusions : Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.

DNA double-strand breaks induced by low energy protons in V79 cells

The initial production of DNA double-strand breaks (dsb) was determined in V79 Chinese hamster cells irradiated with proton beams of 3.24, 1.50 and 0.88 MeV, corresponding to values of unrestricted LET evaluated at the cell midplane of 10.9, 20.0 and 30.5 keV/micron, respectively. X-rays were used for comparison. Dsb were measured with the low speed sedimentation technique in neutral sucrose gradients. The initial yield of dsb rose linearly with the dose and did not significantly depend on the proton LET, in contrast with the results obtained in previous studies for cell inactivation and mutation induction. Also, no significant differences for dsb induction were found between protons and X-rays. Two possible explanations, not necessarily mutually exclusive, are proposed: (1) dsb are not the only lesions involved in cellular effects; and (2) the initial number of dsb is not the only important parameter since a fundamental role is played by the degree of clustering, i.e. the association of dsb with other dsb or other types of damage.

Chromosome Nondisjunction and Loss Induced by Protons and X Rays in Primary Human Fibroblasts: Role of Centromeres in Aneuploidy

Abstract Sgura, A., Cherubini, A. R. and Tanzarella, C. Chromosome Nondisjunction and Loss Induced by Protons and X Rays in Primary Human Fibroblasts: Role of Centromeres in Aneuploidy. Radiat. Res. 156, 225–231 (2001). To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/μm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST−/FISH−, 28.5 keV/μm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST−/FISH+ and CREST+/FISH+, respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/μm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/μm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.